Real-Time PCR

CFX Maestro Dx SE Software
12014330
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Manual revision: October 2022
Software revision: 2.3

User Guide
Version 2.3

CFX Maestro Dx Software, Security
EditionUser Guide
Version 2.3

Bio-Rad Technical Support
The Bio-Rad Technical Support department in the U.S. is open Monday through Friday, 5:00 AM to 5:00 PM,
Pacific Time.
Phone: 1-800-424-6723, option 2
Email: Support@bio-rad.com (U.S./Canada Only)
For technical assistance outside the U.S. and Canada, contact your local technical support office or click theContact us link at www.bio-rad.com.

NoticeNo part of this publication may be reproduced or transmitted in any form or by any means, electronic ormechanical, including photocopy, recording, or any information storage or retrieval system, without permission
in writing from Bio-Rad Laboratories, Inc.
Bio-Rad reserves the right to modify its products and services at any time. This guide is subject to change
without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors or omissions, orfor any damage resulting from the application or use of this information.
BIO-RAD is a trademark of Bio-Rad Laboratories, Inc.
All trademarks used herein are the property of their respective owner.
Copyright © 2022 by Bio-Rad Laboratories, Inc. All rights reserved.

Intended Use

Intended Use
The CFX Opus Dx Real-Time PCR System™ with CFX Maestro Dx Software, Security Edition™ is
intended to perform fluorescence-based PCR to detect and quantitative nucleic acid sequences. The
system and software are intended for in vitro diagnostic use by trained laboratory technicians. The
systems are intended to be used with third-party diagnostic nucleic acid tests, which have been
manufactured and labeled for diagnostic purposes.

Symbols Lexicon

Manufacturer

Lot number

Use byFor In Vitro Diagnostic Use

Temperature limit

Catalog number

Consult instructions for use

Number of tests

For use with

Serial number

User Guide | iii

Prescription use only

Contains latex

CE Marking - Regulation(EU) 2017/746 IVDR

Translations
Product documents may be provided in additional languages on electronic media.

iv | CFX Maestro Dx SE Software

Revision History

Revision History
Document

Date

Description of Change

CFX Maestro Dx Software,

December 2020

Ver A, Initial Release

May 2022

n Updated to support CFX Opus Deepwell Dx

Security Edition User Guide,
2.0(Doc ID #10000134781)
CFX Maestro Dx Software,
Security Edition User Guide,n Updated Symbols Lexicon Table

2.3(Doc ID #10000134781)
CFX Maestro Dx Software,
Security Edition User Guide,

October 2022

n Correction to CFX Opus 384 Dx Ramp Raten Add cybersecurity note to Introduction

2.3(Doc ID #10000134781)

User Guide | v

vi | CFX Maestro Dx SE Software

Table of Contents
Intended Useiii

Symbols Lexiconiii

Translations

iv

Revision Historyv

Safety and Regulatory Compliance17

Safety Warning Labels

17

Safety and Regulatory Compliance19

Safety Compliance

19

Electromagnetic Compatibility (EMC)20

EMC Warnings and Notes

20

Environment Requirements

22

Hazards

23

Biohazards

23

Chemical Hazards

24

Explosive or Flammability Hazards

24

Electrical Hazards

24

Transport

25

Battery25

Disposal

25

Warranty

25

Chapter 1 Introduction

27

Main Features of CFX Maestro Dx Software, Security Edition

28

Finding Out More

28

Chapter 2 Installing CFX Maestro Dx Software, Security Edition

29

System Requirements

30

Installing CFX Maestro Dx SE Software

31

Detecting Connected Instruments

32

Software Files

33

User Guide | vii

Table of Contents

Chapter 3 Managing CFX Maestro Dx Software, Security Edition User Accounts

35

Starting CFX Maestro Dx Software, Security Edition

36

Adding Microsoft Windows Users to the CFX Maestro Dx Software, Security Edition Computer

38

Adding and Removing CFX Maestro Dx Software, Security Edition Users

40

Managing CFX Maestro Dx Software, Security Edition User Roles

41

Viewing Your Role and Permissions

Chapter 4 Using CFX Maestro Dx Software, Security Edition
Secure Files

Chapter 5 The Workspace

42

43
43

53

The Home Window

54

The Startup Wizard

55

The Protocol Editor Window

56

The Plate Editor Window

57

The Data Analysis Window

58

Chapter 6 The Home Window

59

The Home Window

60

File Menu Commands

61

View Menu Commands

61

User Menu Commands

62

Run Menu Commands

62

Tools Menu Commands

63

Help Menu Commands

63

Toolbar Commands

64

The Startup Wizard

65

Status Bar

65

Detected Instruments Pane66

Viewing the Properties of an Instrument

69

Before You Begin

70

Creating a Reaction Master Mix

70

Calibrating New Dyes

72

Setting User Preferences

75

Chapter 7 Creating Protocols

91

Parameters and Ranges for Protocol Steps

viii | CFX Maestro Dx SE Software

92

Table of Contents

Protocol Editor Window

94

File Menu Commands

94

Settings Menu Command95

Tools Menu Commands

95

Toolbar Commands

95

Protocol Editing Controls

96

Creating a Protocol in the Protocol Editor

98

Opening a New Protocol File in the Protocol Editor

98

Opening an Existing Protocol in the Protocol Editor

99

Setting Up a New Protocol

100

Adding Steps to a Protocol

102

Inserting a Gradient Step

103

Inserting a GOTO Step

104

Inserting a Melt Curve Step

104

Adding or Removing a Plate Read Step

106

Changing Step Options

106

Deleting a Step107

Copying, Exporting, or Printing a Protocol

107

Creating a Protocol with the Protocol AutoWriter

108

Using the Ta Calculator

110

About the Ta Calculator

110

Chapter 8 Preparing Plates

115

Plate Editor Window

116

File Menu Commands

116

Edit Menu Commands

117

Settings Menu Commands

117

Editing Tools Menu Commands

117

Toolbar Commands

118

Creating a Plate File Using the Plate Editor

119

Opening a New Plate File in the Plate Editor

119

Opening an Existing Plate File in the Plate Editor

121

Setting Up a New Plate File

122

Assigning Optional Parameters to the Plate File
Assigning a Target to Wells

129
129

User Guide | ix

Table of Contents

Assigning a Sample Name to Wells

131

Assigning Biological Groups to Wells

133

Assigning Technical Replicate Numbers to Wells

134

Assigning a Dilution Series to Standard Sample Types

136

Copying Well Contents into Another Well137

Adding a Note to a Well

138

Clearing Wells of All Content

138

Changing Experiment Settings

140

Creating Well Groups

143

Changing Trace Styles

145

Viewing, Exporting, and Importing the Plate in Spreadsheet Format

146

Creating a Plate Layout Using the Plate Setup Wizard

148

Using the Plate Setup Wizard

148

Chapter 9 Running Experiments

151

The Run Setup Window
Accessing the Run Setup Window

152
153

Protocol Tab

154

Plate Tab

156

Start Run Tab

158

Running an Experiment

159

Run Details Dialog Box

161

Run Status Tab

161

Real-time Status Tab

164

Time Status Tab

167

Performing PrimePCR Experiments

168

Transferring Stand-Alone Data for Analysis

170

Transferring Data Through Email170

Transferring Data from CFX Opus Dx Real-Time PCR Systems

170

Transferring Data through CFX Maestro Dx Software, Security Edition

173

Transferring Data Using a USB Drive

173

Transferring Data through a Shared Network Drive Using CFX Opus Dx Real-Time PCR
Systems

174

Creating a Data File174

x | CFX Maestro Dx SE Software

Table of Contents

Chapter 10 Data Analysis Overview
Data Analysis Window

175
175

Data Analysis Toolbar

176

Data Analysis Menu Bar177

Tab Details

179

Step Number Selector

180

Viewing Well Groups in Data Analysis

181

Changing Well Contents after a Run

181

Data Analysis Settings

182

Adjusting the Threshold

182

Baseline Settings

182

Analysis Mode

183

Cycles to Analyze

184

Well Selector

185

Well Selector Right-Click Menu Items

186

Temporarily Excluding Wells from Analysis

187

Charts188

Chart Tools

188

Magnifying an Area in the Chart

196

Copying Charts into a Microsoft File

196

Common Right-Click Menu Items for Charts

196

Spreadsheets
Common Right-Click Menu Items for Spreadsheets
Export

197
197
199

Exporting All Data Sheets

199

Exporting RDML Files

200

Creating a Custom Export File

201

Exporting to a LIMS Folder

202

Exporting Seegene-Formatted Data

202

Chapter 11 Data Analysis Details
Quantification Tab

203
204

Fluorophore Options

204

Trace Styles Dialog Box

205

Log Scale Option

206

User Guide | xi

Table of Contents

Standard Curve Chart

207

Amplification Chart Menu Options

208

Quantification Tab Spreadsheet

208

Quantification Data Tab

210

Results Spreadsheet

210

Standard Curve Results Spreadsheet

212

Plate Spreadsheet

213

RFU Spreadsheet

214

Melt Curve Tab
Adjusting Melt Curve Data
Melt Curve Data Tab

215
217
218

Melt Peaks Spreadsheet

218

Plate Spreadsheet

219

RFU Spreadsheet

220-d(RFU)/dT Spreadsheet

221

End Point Tab

222

Results Data

223

Adjusting the End Point Data Analysis

224

RFU Spreadsheet for End Point Analysis

224

Allelic Discrimination Tab

225

Adjusting Data for Allelic Discrimination

226

Chart Menu Options

227

Allelic Discrimination Spreadsheet

227

Custom Data View Tab
Creating a Custom Data View
QC Tab

228
229
230

Changing QC Criteria

230

Excluding Wells That Fail QC

231

Run Information Tab

232

Data Analysis Reports

233

Data Analysis Report Categories

234

Creating a Data Analysis Report

237

Creating Well Group Reports

238

xii | CFX Maestro Dx SE Software

Table of Contents

Chapter 12 Gene Expression Analysis

239

Plate Setup for Gene Expression Analysis

239

Guided Plate Setup

240

Gene Expression Charts

241

Graphing

242

Changing and Annotating the Chart View

244

Adjusting Gene Expression Data

249

Experiment Settings

251

Right-Click Menu Options

252

Data Spreadsheet

253

Show Details Option

254

Clustergram

256

Settings

256

Right-Click Menu Options

256

Data Spreadsheet

256

Scatter Plot

257

Settings

257

Right-Click Menu Options

257

Data Spreadsheet

257

Results Spreadsheet

258

Gene Study

259

Inter-Run Calibration

259

Gene Study Dialog Box

260

Study Setup Tab

260

Preparing a Gene Study

261

Study Analysis Tab

262

Gene Study Report Categories

263

Creating a Gene Study Report

265

Appendix A Data Analysis Calculations

267

Reaction Efficiency

267

Relative Quantity

267

Relative Quantity When a Control Is Selected

268Standard Deviation of Relative Quantity

268

Efficiency Corrected Cq (CqE)

269

User Guide | xiii

Table of Contents

Mean Efficiency Corrected Cq (MCqE)

269

Normalized Expression

270

Expression and Relative Quantity for Biological Groups

271

Normalized Expression When a Control Is Selected271Standard Deviation for the Normalized Expression

272

Normalized Expression Scaled to Highest Expression Level

273

Normalized Expression Scaled to Lowest Expression Level

273

Normalized Expression Scaled to Average Expression Level

273

Standard Deviation for the Scaled Normalized Expression

274

Error Bars for Standard Deviation(lg) and Standard Error of the Mean (lg)275

Fold Change

276

Corrected Values Formulas

277

Confidence Interval Calculation for Biological Group Analysis

278

Box and Whisker Chart Calculations

278

Appendix B Audit Trails

281

Viewing Audit Trails

281

Auditable Events

283

Appendix C LIMS Integration
Creating LIMS-Compatible Data Files

287
287

Setting Up LIMS Folder and Data Export Options

287

Creating a LIMS Protocol

289

Creating a LIMS File

289

Starting a LIMS Run

292

Exporting Data to a LIMS

293

Appendix D Troubleshooting CFX Maestro Dx Software, Security Edition

295

Whitelist CFX Maestro Dx Software, Security Edition Files and Folders

295

Application Log

296

Retrieving Application and Firmware Log Files

297

Troubleshooting

297Power Failure

297

Transferring Files to the CFX Maestro Dx SE Computer

298

Installing CFX Maestro Dx Software, Security Edition Manually

298

Reinstalling the Drivers

298

xiv | CFX Maestro Dx SE Software

Table of Contents

Appendix E Bio-Rad Free and Open-Source Notices for PCR Products

301

Software Notices

302

ZedGraph

302

Standard Open License Text
LGPL-2.1

Appendix F References

302
302

315

User Guide | xv

Table of Contents

xvi | CFX Maestro Dx SE SoftwareSafety and Regulatory Compliance
The CFX Opus 96 Dx, CFX Opus 384 Dx, and CFX Opus Deepwell Dx Real-Time PCR systems (known
in this guide as the CFX Opus Dx system) heat and cool very quickly during operation. For safe operationof the real-time PCR system, Bio-Rad strongly recommends that you follow the safety specifications listed
in this section and throughout this manual.

Safety Warning Labels
Warning labels posted on the CFX Opus Dx system and in this manual inform you of sources of injury or
harm. Table 1 defines each safety warning label.
Table 1. General safety warnings

Icon

Meaning
Operating the CFX Opus Dx system before reading this manual can constitute a
personal injury hazard. The use of this instrument in a manner not specified in thismanual or by Bio-Rad may result in the protection features of the instrument
becoming impaired or disabled.
There are no biohazards or radioactive hazards associated with the CFX Opus Dx
system itself. These hazards only become a concern when they are introduced into
the system via the samples being tested. When handling biohazardous orradioactive samples, adhere to the recommended precaution s and guidelines
specific to your laboratory and location. These guidelines should include cleaning,monitoring, and disposal methods for the hazardous materials you are using.
In addition, as identified above, there is a small risk of explosion, or of expulsion ofliquids or vapors from the sample containers. When working with hazardous
materials the risk of injury from expelled material is compounded with the risk thatthe hazardous material themselves could be dispersed in and around the
instrument. Users should take appropriate precautions for such a situation.

User Guide | 17

Safety and Regulatory Compliance

Table 1. General safety warnings, continued

Icon

Meaning
The CFX Opus Dx systems operate at temperatures high enough to cause serious
burns. Always allow the sample block to return to room temperature before opening
the lid and removing samples. Even after the sample block has cooled, thesurrounding areas as well as the heater plate can remain hot for quite some time. In
situations where there is not sufficient time to allow the instrument to cool, the useof protective equipment such as thermal gloves or “oven mitts” is recommended.
The safety and performance of any system incorporating a CFX Opus Dx system issolely the responsibility of the assembler of the system.

The CFX Opus Dx system can become hot enough during the course of normal
operation to cause liquids in the samples to boil or vaporize, pressurizing thesample containers. There is the possibility that the sample containers could fail,leading to leaks, fluid spray, or explosive rupture and expelling vapors or liquids inand around the instrument.
Users should always operate the instrument with the lid closed or wear safety
goggles, thermal gloves, and other personal protection equipment during operation
to avoid injury. Opening the instrument while samples are still hot, such as after
aborting a run, can allow pressurized containers to leak, spray, or spurt liquid.
Always allow the samples to cool before opening the lid.
Users should never run a reaction with a lid or seal that is open, loose, punctured,or otherwise damaged because it will increase the likelihood of a dangerous
rupture or explosion.
Users should never run a reaction with volatile reagents that could increase thelikelihood of a dangerous rupture or explosion.

18 | CFX Maestro Dx SE SoftwareSafety and Regulatory Compliance

Safety and Regulatory Compliance
Safety Compliancen

IEC 61010-1:2010 Safety requirements for electrical equipment for measurement, control, andlaboratory use, Part 1: General requirementsn

IEC 61010-2-010:2019 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-010: Particular requirements for laboratory equipment for the heating ofmaterialsn

IEC 61010-2-081:2019 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-081: Particular requirements for automatic and semi-automatic laboratory
equipment for analysis and other purposesn

IEC 61010-2-101:2018 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipmentn

CAN/CSA-C22.2 NO. 61010-1-12:2018 Safety requirements for electrical equipment formeasurement, control, and laboratory use, Part 1: General Requirementsn

CAN/CSA-C22.2 NO. 61010-2-010:19 Safety requirements for electrical equipment formeasurement, control, and laboratory use, Part 2-010: Particular requirements for laboratory
equipment for the heating of materialsn

CAN/CSA-C22.2 NO. 61010-2-081:19 Safety requirements for electrical equipment formeasurement, control, and laboratory use, Part 2-081: Particular requirements for automatic andsemi-automatic laboratory equipment for analysis and other purposesn

CSA-C22.2 NO. 61010-2-101:19 Safety requirements for electrical equipment for measurement,
control and laboratory use — Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical
equipmentn

EN 61010-1:2010 Safety requirements for electrical equipment for measurement, control, andlaboratory use, Part 1: General requirementsn

EN 61010-2-010:2014 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-010: Particular requirements for laboratory equipment for the heating ofmaterials

User Guide | 19

Safety and Regulatory Compliancen

EN 61010-2-081:2015 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-081: Particular requirements for automatic and semi-automatic laboratory
equipment for analysis and other purposesn

EN 61010-2-101:2017 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipmentn

UL 61010-1:2012 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 1: General Requirementsn

UL 61010-2-010:2019 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-010: Particular requirements for laboratory equipment for the heating ofmaterialsn

UL 61010-2-081:2019 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-081: Particular requirements for automatic and semi-automatic laboratory
equipment for analysis and other purposesn

UL 61010-2-101:19 Safety requirements for electrical equipment for measurement, control andlaboratory use — Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipment

Electromagnetic Compatibility (EMC)n

IEC 61326-1:2020 Electrical equipment for measurement, control and laboratory use — EMC
requirements — Part 1: General requirements. Tested as a Class A devicen

IEC 61326-2-6:2012 Electrical equipment for measurement, control and laboratory use — EMC
requirements — Part 2–6: Particular requirements – In vitro diagnostic (IVD) medical equipmentn

EN 61326-1:2013 Electrical equipment for measurement, control and laboratory use — EMC
requirements — Part 1: General requirements. Tested as a Class A devicen

EN 61326-2:-6:2013 Electrical equipment for measurement, control and laboratory use — EMC
requirements — Part 2–6: Particular requirements – In vitro diagnostic (IVD) medical equipmentn

FCC Part 15, Subpart B, Sections 15.107 and 15.109. Tested as a Class A digital devicen

CAN ICES-003v6:2019 Interference-causing equipment standard, information technology equipment(including digital apparatus) — Limits and methods of measurement. Tested to Class A limits

EMC Warnings and NotesnWarning: Changes or modifications to this unit, not expressly approved by Bio-Rad, could void theuser’s authority to operate the equipment.

20 | CFX Maestro Dx SE SoftwareSafety and Regulatory CompliancenNote: This equipment has been tested and found to comply with the limits for a Class A digital device,pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection
against harmful interference when the equipment is operated in a commercial environment. Thisequipment generates, uses, and can radiate radio frequency energy and, if not installed and used inaccordance with the instruction manual, may cause harmful interference to radio communications.
Operation of this equipment in a residential area is likely to cause harmful interference, in which casethe user will be required to correct the interference at their own expense.

nNote regarding FCC compliance: Although this instrument has been tested and found to comply
with Part 15, Subpart B of the FCC Rules for a Class A digital device, please note that thiscompliance is voluntary, for the instrument qualifies as an “exempted device” under 47 CFR 15.103(c), in regard to the cited FCC regulations in effect at the time of manufacture.

nNote regarding cables: This instrument was tested for EMC compliance using specially designed
USB cables, which are supplied with the instrument. These cables, or Bio-Rad authorized
replacements, must be used with this instrument to ensure continued compliance with the EMC
emissions limits.

User Guide | 21

Safety and Regulatory Compliance

Environment Requirements
The CFX Opus Dx system has been designed to be safely operated under the environmental conditions
listed in the following table.
Table 2. CFX Opus Dx Real-Time PCR System environment requirements

Parameter

Specification

Environment

Indoor use only

Operating altitude

Up to 2,000 meters above sea level

Ambient room temperature

15–31°C*
59–88°F

Transport and storage temperature–20° to 60°C**–4 to 140°F

Relative humidity

20% to 80% (noncondensing)***Operating power

100 to 240 VAC, 50/60 Hz, 850 W Max

Mains supply voltage fluctuation±10%

Maximum power usage<850 watts

Fuses

10 A, 250 V, 5 x 20 mm, fast blow (qty. 2)

Overvoltage category

II

Pollution degree2* Operating the instrument outside of this temperature range may not meet performance specifications.
A room temperature between 5–40°C (41–104°F) is considered safe.
** Store and transport the instrument in its shipping container to meet these temperature conditions.
*** Operating the instrument at 4⁰C should be limited to 18 hours at these conditions. Holds at 4⁰C canbe performed for up to 72 hours if humidity is 20% to 60% (noncondensing).

22 | CFX Maestro Dx SE Software

Hazards

Hazards
The CFX Opus Dx system is designed to operate safely when used in the manner prescribed by the
manufacturer. If thesystem or any of its associated components is used in a manner not specified by the
manufacturer, the inherent protection provided by the instrument may be impaired. Bio-Rad is not liable
for any injury or damage caused by the use of this equipment in any unspecified manner, or bymodifications to the instrument not performed by Bio-Rad or an authorized agent. Service of the CFX
Opus Dx system should be performed only by trained Bio-Rad personnel.

Biohazards
The CFX Opus Dx system is a laboratory product. However, if biohazardous samples are present, adhere
to the following guidelines and comply with any local guidelines specific to your laboratory and location.
Note: No biohazardous substances are exhausted during normal operations of this instrument.

General Precautionsn

Always wear a laboratory coat, laboratory gloves, and safety glasses with side shields or goggles.

n

Keep your hands away from your mouth, nose, and eyes.

nCompletely protect any cut or abrasion before working with potentially infectious materials.

nWash your hands thoroughly with soap and water after working with any potentially infectious
material before leaving the laboratory.

n

Remove wristwatches and jewelry before working at the bench.

nStore all infectious or potentially infectious material in unbreakable leak-proof containers.

nBefore leaving the laboratory, remove protective clothing.

n

Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch anything that
other people may touch without gloves.

n

Change gloves frequently. Remove gloves immediately when they are visibly contaminated.

n

Do not expose materials that cannot be properly decontaminated to potentially infectious material.

nUpon completion of an operation involving biohazardous material, decontaminate the work area with
an appropriate disinfectant (for example, a 1:10 dilution of household bleach).

User Guide | 23

Safety and Regulatory Compliance

Surface Decontamination
WARNING! To prevent electrical shock, always turn off and unplug the instrument prior
to performing decontamination procedures.

The following areas can be cleaned with any hospital-grade bactericide, virucide, or fungicide disinfectant:nOuter lid and chassisn

Inner sample block surface and sample block wellsnControl panel and display

To prepare and apply the disinfectant, refer to the instructions provided by the product manufacturer.
Always rinse the sample block and sample block wells several times with water after applying adisinfectant. Thoroughly dry the sample block and sample block wells after rinsing with water.
Important: Do not use abrasive or corrosive detergents or strong alkaline solutions. These agentscan scratch surfaces and damage the sample block, resulting in loss of precise thermal control.

Disposal of Biohazardous Material
Dispose of the following potentially contaminated materials in accordance with laboratory local, regional,
and national regulations:nClinical samples

nReagents

n

Used reaction vessels or other consumables that may be contaminatedChemical Hazards
The CFX Opus Dx system contains no potentially hazardous chemical materials.

Explosive or Flammability Hazards
The CFX Opus Dx system poses no uncommon hazard related to flammability or explosion when used ina proper manner as specified by Bio-Rad Laboratories.

Electrical Hazards
The CFX Opus Dx system poses no uncommon electrical hazard to operators if installed and operated
properly without physical modification and connected to a power source of proper specification.

24 | CFX Maestro Dx SE Software

Transport

Transport
Before moving or shipping the CFX Opus Dx system, decontamination procedures must be performed.
Always move or ship the system in a separate container in the Bio-Rad-supplied packaging material,
which protects the system from damage.
For information about transporting the system and to request the appropriate packaging material, contact
your local Bio-Rad office.

Battery
The CFX Opus Dx system uses one 3 V lithium-metal coin cell battery to maintain time settings in theevent of AC power loss. If the time does not remain set after the unit is turned off, it may be an indicationthat the batteries are getting weak.
WARNING! Do not attempt to change the batteries. They are not user serviceable.
Instead, contact Bio-Rad Technical Support for assistance.

For the State of California, USA onlynPerchlorate material — Lithium batteries contain perchlorate material; special handling may apply.
See www.dtsc.ca.gov/hazardouswaste/perchlorate.

Disposal
The CFX Opus Dx systems contain electrical materials; they should not be disposed of as unsorted waste
and must be collected separately, according to European Union Directive 2012/19/EU on waste electricaland electronic equipment — WEEE Directive. Before disposal, contact your local Bio-Rad representative
for country-specific instructions.

Warranty
The CFX Opus Dx system and its associated accessories are covered by a standard Bio-Rad warranty.
Contact your local Bio-Rad office for the details of the warranty.

User Guide | 25

Safety and Regulatory Compliance

26 | CFX Maestro Dx SE SoftwareChapter 1 Introduction
Bio-Rad’s high-performance PCR amplification systems feature the latest technological advances,providing greater accuracy and reproducibility in nucleic acid amplification for genomic experiments.
Bio-Rad’s CFX Maestro Dx Software, Security Edition is compatible with the following instruments andfeatures optimized run files for Bio-Rad’s PrimePCR primer and probe assays:n

CFX Opus 96 Dx Real-Time PCR System (known in this guide as CFX Opus 96 Dx)n

CFX Opus 384 Dx Real-Time PCR System (known in this guide as CFX Opus 384 Dx)n

CFX Opus Deepwell Dx Real-Time PCR System (known in this guide as CFX Opus Deepwell Dx)

Using CFX Maestro Dx Software, Security Edition (known in this guide as CFX Maestro Dx SE) you caninterpret complex data and craft powerful studies for genetic analysis. With just a few clicks, you can set
up studies and make sense of your gene expression study with tools such as t-tests, one-way ANOVA,
PrimePCR controls analysis, and the reference gene selector tool. Then, you can prepare your results for
publications and posters with CFX Maestro Dx SE’s highly customizable data visualization and annotation
tools.
Note: The display of some screens in CFX Maestro might appear different than those represented inthis user guide. The display in the software is correct, and the functionality is the same.
Important: Cybersecurity is the protection of assets in cyberspace from cyberattacks. Cybersecurity
is Bio-Rad’s ability to secure its people, information, systems, and reputation in cyberspace.
Cyberspace is the always-on, technologically interconnected world; it consists of people,
organizations, information, and technology.
Fast reaction is important with cybersecurity issues! If you suspect that there may be a cybersecurity
issue concerning your instrument or that cybersecurity has been breached at your site, contact your
Bio-Rad representative for technical support immediately.

User Guide | 27

Chapter 1 Introduction

Main Features of CFX Maestro Dx Software, Security Edition
With CFX Maestro Dx SE you can do the following:nAnalyze data using bar charts, clustergrams, or scattergrams to quickly interpret and understandyour results.

nCustomize your data representation and export high resolution graphs for publication and report
generation.

n

Determine RNA quality and troubleshoot experiments with PrimePCR analysis controls.

nSelect the appropriate reference gene and analyze its stability with the Reference Gene selection
tool.

n

Perform statistical analysis including one-way ANOVA in gene expression analysis.

This user guide explains these features and how to use them.

Finding Out More
After installing CFX Maestro Dx SE and setting up the associated Bio-Rad PCR instrument, you canaccess this guide as well as detailed CFX Maestro Dx SE help topics from the Help menu in any view.
Tip: Click the Bio-Rad logo in the upper right corner of any CFX Maestro Dx SE window to launch
Bio-Rad’s website. This site includes links to technical notes, manuals, videos, product information,and technical support. This site also provides many technical resources on a wide variety of methods
and applications related to PCR, real-time PCR, and gene expression.

28 | CFX Maestro Dx SE SoftwareChapter 2 Installing CFX Maestro Dx Software,
Security Edition
This chapter explains how to install CFX Maestro Dx Software, Security Edition. For information about
setting up Bio-Rad's supported real-time PCR instruments, see the appropriate guide.
CFX Maestro Dx SE is required to analyze real-time PCR data from the CFX Opus 96 Dx, CFX Opus 384
Dx, and CFX Opus Deepwell Dx Real-Time PCR systems. You can also use this software to control thesesystems in software-controlled mode.
CFX Opus Dx systems ship with a USB cable in the Accessory bag. Use the USB cable to connect thecomputer running CFX Maestro Dx SE to the CFX Opus Dx system.
Remove the packing materials and store them for future use. If an item is missing or damaged, contact
your local Bio-Rad office.

User Guide | 29

Chapter 2 Installing CFX Maestro Dx Software, Security Edition

System Requirements
Table 3 lists the minimum and recommended system requirements for the computer running the
CFX Maestro Dx SE.
Table 3. Computer requirements for CFX Maestro Dx SE

SystemMinimum

Recommended

OperatingMicrosoft Windows 10 (64-bit

Microsoft Windows 10 (64-bit only), build 1511

systemonly), build 1511 or later, with theor later, with the latest security updates.

latest security updates.
Note: Windows 11 also supports CFX Maestro Dx Software, Security Edition.
Important: Secure Boot must be disabled on computers running CFX Maestro Dx SE. Computers
running CFX Maestro Dx SE should be configured so that they do not automatically restart after asystem or security update if a run is in progress. See your system administrator for assistance.
Ports

2 USB 2.0 High-speed ports

2 USB 2.0 High-speed ports

Hard disk space

128 GB

128 GB

Processor speed

2.4 GHz, Dual Core

2.4 GHz, Quad CoreRAM

4 GB RAM

8 GB RAM

Screen1024 x 768 with true-color mode

1280 x 1024 with true-color mode

resolution
PDF reader

Adobe PDF Reader or Windows PDF Reader
from one of the supported Microsoft Office
Suites:n 2016n 2019Localization

Supported Microsoft Windows

Supported Microsoft Windows 64-bit OS in

64-bit OS in English, Chinese,

English, Chinese, and Russianand Russian

Note: If you plan to run CFX Automation Control software on the same computer as CFX Maestro Dx
SE, set the screen resolution to 1280 x 1024 with true-color mode.

30 | CFX Maestro Dx SE SoftwareInstalling CFX Maestro Dx SE Software

Installing CFX Maestro Dx SE Software
Important: You must disconnect any connected instruments from the CFX Maestro Dx SE computer
before you install or upgrade the software. You do not need to turn off the instrument during thesoftware installation. Ensure that you have saved all runs and that no experiments are running.
Note: Verify that Secure Boot is disabled before beginning the installation procedure. Ensure that thecomputer is configured such that it does not automatically restart after a system or security update if a
run is in progress. See your system administrator for assistance.

To install CFX Maestro Dx SE software1.

If necessary, disconnect any connected instruments from the computer.
Locate and disconnect the instrument’s USB cable on the CFX Maestro Dx SE computer. The end
inserted in the CFX Opus Dx system can remain in place.

2.

Log in to the CFX Maestro Dx SE computer with administrative privileges.

3.

Insert the CFX Maestro Dx SE software USB drive into the computer’s USB port.

4.

In Windows Explorer, navigate to and open the CFX Maestro Dx SE software USB drive.
The USB drive contains the Release Notes and the following folders:
n

CFX

n

Driversn

Firmwaren

Quick Start
Along with other files, the CFX folder contains the CFX Maestro Dx SE software installer(CFXMaestroDxSetup.exe).

5.

Open the CFX folder and double-click CFXMaestroDxSetup.exe to start the installer.

6.

Follow the on-screen installation instructions.
When completed, the Bio-Rad CFX Maestro Dx Software, Security Edition icon appears on thecomputer desktop.
Tip: The CFX Maestro installer automatically installs the CFX Maestro Dx Software, Security
Edition user guide. To locate these guides, navigate to the Help menu and select Open User
Guides.

7.

After the installation completes, you can safely eject the software USB drive.

User Guide | 31

Chapter 2 Installing CFX Maestro Dx Software, Security Edition

Detecting Connected Instruments
During installation, the CFX Maestro Dx SE installer automatically installs the instrument drivers onto the
CFX Maestro Dx SE computer. CFX Maestro Dx SE detects connected instruments when you start thesoftware.

To detect connected instruments
1.

If you have not yet done so, insert the square (male) end of the supplied USB Type B cable into the
USB Type B port located on the back of the instrument’s base.

2.

Insert the other (port) end into a USB port on the CFX Maestro Dx SE computer.

3.

If the instrument is not already running, press the power switch on the instrument to turn it on.

4.

Start CFX Maestro Dx SE.

The software automatically detects the connected instrument and displays its name in the Detected
Instruments pane on the Home window.
Note: If the instrument does not appear in the Detected Instruments pane, verify that the USB cableis properly installed. To reinstall drivers, on the Home window in CFX Maestro Dx SE selectTools > Reinstall Instrument Drivers.

32 | CFX Maestro Dx SE Software

Software Files

Software Files
Table 4 lists the CFX Maestro Dx SE file types.
Table 4. CFX Maestro Dx SE file types

File Type

Extension

Details

Protocol.prcl

Contains protocol setup details to perform a PCR run.

Plate

.pltd

Contains plate setup details to perform a PCR run.

Data

.pcrd

Contains the results of an experiment run and PCR
analysis.

PrimePCR run.csv

Contains protocol and plate layout for PrimePCR plates.

Gene study.mgxd

Contains results of multiple PCR runs and gene expression
analyses.

Stand-alone pre-data

.zpcr

file
LIMS

Contains fluorescence readings from stand-alone
operation that are converted into a data file.

.plrn

Contains plate setup and protocol information required to
conduct a LIMS compatible run.

JSON

.json

A read-only file generated only by CFX Opus Dx systems,
this file contains the run file data that appear in the details
pane in the File Browser when a run file is selected. This
file is generated after a run completes. It is exported withthe .zpcr file and saved with the data files when the Save
Location is either a USB drive or a shared network folder.

User Guide | 33

Chapter 2 Installing CFX Maestro Dx Software, Security Edition

34 | CFX Maestro Dx SE SoftwareChapter 3 Managing CFX Maestro Dx Software,
Security Edition User Accounts
In CFX Maestro Dx Software, Security Edition, users log in with their Windows user name and password.
The person who installed CFX Maestro Dx SE is automatically assigned the role of Administrator and cancreate and manage user accounts and roles. All other users must be assigned a user account in order tolog into and use the software.
Important: Each user must have a Windows account and password on the CFX Maestro Dx SE
computer before you can assign a user account and role. Users can be members of either the
Windows Users group or Windows Administrators group. Members of the Windows Users group canaccess only their own CFX Maestro Dx SE files and folders. Members of the Windows Administratorsgroup can access the files and folders of all users on the computer.
This chapter explains how to create Microsoft Windows users in order to add those users to CFX Maestro
Dx SE. This section also explains how to add CFX Maestro Dx SE users and manage user roles andpermissions.

User Guide | 35

Chapter 3 Managing CFX Maestro Dx Software, Security Edition User Accounts

Starting CFX Maestro Dx Software, Security Edition
Note: Each user must log in with their Windows user name and password.

To start CFX Maestro Dx SE
1.

On the CFX Maestro Dx SE computer desktop, double-click the CFX Maestro Dx SE shortcut icon tostart the application.

2.

In the Login dialog box, type your Windows password and click OK.

CFX Maestro Dx SE opens to the Home window. The title bar displays the Windows user name of thelogged in user, and the menu bar displays a blue sticker indicating the software is 21 CFR Part 11
compliant, for example:

36 | CFX Maestro Dx SE Software

Starting CFX Maestro Dx Software, Security Edition

User Guide | 37

Chapter 3 Managing CFX Maestro Dx Software, Security Edition User Accounts

Adding Microsoft Windows Users to the CFX Maestro Dx
Software, Security Edition Computer
All users must log in to the CFX Maestro Dx SE computer with their Windows user name and password.
For accurate audit-tracking, Windows user accounts cannot be added via the Start > Settings > Accounts
dialog box. Windows user accounts must be added via the Computer Management console.
Important: Changes made to Windows user properties (including the User name and Full name)after you have created the associated CFX Maestro Dx SE user invalidates the CFX Maestro Dx SE
user. Ensure the information is correct before saving the Windows user and creating the associated
CFX Maestro Dx SE user.
Tip: Review the Microsoft Windows Administration documentation and see your Windows systemadministrator for more information before creating Windows accounts.

To add Windows user accounts to the CFX Maestro Dx SE computer1.

Log on to the CFX Maestro Dx SE computer as a member of the Windows Administrator’s group.

2.

On the desktop, right-click My Computer and select Manage to open the Computer Management
console.

3.

In the Computer Management console, expand Local Users and Groups.

4.

Right-click the Users folder and select New User to open the New User dialog box.

38 | CFX Maestro Dx SE SoftwareAdding Microsoft Windows Users to the CFX Maestro Dx Software, Security Edition Computer

5.

6.

In the New User dialog box, you must complete the following fields:nUser namen

Full namenPasswordnConfirm password

Click Create.

User Guide | 39

Chapter 3 Managing CFX Maestro Dx Software, Security Edition User Accounts

Adding and Removing CFX Maestro Dx Software, Security
Edition Users
Tip: Only users with the CFX Maestro Dx SE Administrator role can create and remove CFX Maestro
Dx SE user accounts. The person who installed CFX Maestro Dx SE is automatically assigned theAdministrator role. That person can assign the Administrator role to other users.
Note: In CFX Maestro Dx SE, at least one user must be assigned the Administrator role.

To add CFX Maestro Dx SE user accounts
1.

Verify that each intended user is a member of either the Windows Users group or Windows
Administrators group and has a Windows password on the CFX Maestro Dx SE computer.

2.

Start CFX Maestro Dx SE and log in as the Administrator.

3.

In the Home window, select User > User Administration.
The User Administration dialog box appears.

40 | CFX Maestro Dx SE Software

Managing CFX Maestro Dx Software, Security Edition User Roles

4.

In the Manage Users section, provide the following information for each user:nUser name — in CFX Maestro Dx SE, this must be the user’s Windows login user name.

n

Full name — the user’s full name.
This name appears in the Full User field in the audit trail. This name must be the same nameentered in the Full Name field when the Windows user was created.

nRole — the role to assign to the user.
Note: You can select only one role from the dropdown list. See Managing CFX Maestro Dx
Software, Security Edition User Roles for more information.

n

Domain — the Windows domain from which the user accesses the software.
See your Windows system administrator for more information.

5.

Click OK and then click Yes to save changes and close the User Administration dialog box.

To remove a CFX Maestro Dx SE user account1.

Start CFX Maestro Dx SE and log in as the Administrator.

2.

In the Home window, select User > User Administration to open the User Administration dialog box.

3.

In the Manage Users pane, select Remove for each user you want to remove.

4.

Click OK and then click Yes to save changes and close the User Administration dialog box.

Managing CFX Maestro Dx Software, Security Edition User
Roles
Important: CFX Maestro Dx SE requires that at least one user be assigned the Administrator role.
You can assign this role to more than one user.
CFX Maestro Dx SE has four user roles. Each user must be assigned a role in order to access thesoftware. Although users can be assigned only one role, you can change a user’s role at any time.
Except for the Administrator role, you can change the permissions assigned to each role. All users
assigned a role inherit only the permissions of that role.
By default, the rights for each role are as follows:nAdministrator — this role has all permissions; you cannot change these permissions.

nPrincipal — this role has all permissions except to set up email.

n

Operator — this role has all permissions except to skip cycles and set up email.

User Guide | 41

Chapter 3 Managing CFX Maestro Dx Software, Security Edition User AccountsnGuest — this role can only read files.

When assigning roles in CFX Maestro Dx SE, carefully determine the requirements for each user. For
example, without the permission to save, users assigned the Guest role will not be able to sign a file.
Without the permission to set up an email account, none of the roles will receive email when a run
completes.

To modify the permissions for a role
1.

Start CFX Maestro Dx SE and log in as the Administrator.

2.

In the Home window, select User > User Administration to open the User Administration dialog box.

3.

In the Manage Rights section, for each role clear or select the checkbox of specific permissions asnecessary.

4.

Click OK and then click Yes to save changes and close the User Administration dialog box.

Viewing Your Role and Permissions
Tip: Users assigned the Principal, Operator, or Guest user roles can view only their user settings,
permissions, and roles. Users assigned the Administrator role can view all user permissions androles.

To view your current user role and permissionsu

In the Home window, select User > User Administration.

Contact your CFX Maestro Dx SE administrator to modify the user settings, permissions, and roles listed
in the User Administration window.

42 | CFX Maestro Dx SE SoftwareChapter 4 Using CFX Maestro Dx Software,
Security Edition
Important: CFX Maestro Dx Software, Security Edition uses Microsoft Windows User Authentication
to verify access to secure CFX data files. Contact your Windows administrator to create anenvironment that complies with 21 CFR Part 11 requirements.
Using CFX Maestro Dx SE, users cannSign data and gene study files.

n

Password protect data files.

nView and print audit trails.

This section explains these features in detail.

Secure Files
By default, CFX Maestro Dx SE saves secure files to the logged in user’s personal folder, which is locatedat
C:\Users\<username>\Documents\Bio-Rad\CFX_MDX\My qPCR
You can save and edit .pcrd files in that folder. This folder contains links to other folders (for example, theSample Files Folder) that contain files that are read-only. However, an administrator can delete thecontents of that folder.
Tip: Alternatively, your Windows system administrator can create a shared folder and your
CFX Maestro Dx SE administrator can program the software to save all files to that folder.
In CFX Maestro Dx SE the plate, protocol, data, and gene study files are marked as secure when they are
saved. You can create these files in CFX Maestro software or in CFX Maestro Dx SE. After they are savedin CFX Maestro Dx SE, you can open these files only in CFX Maestro Dx SE.
CFX Maestro Dx SE creates an audit trail for all secure data and gene study files (.pcrd and .mgxd files,
respectively). The software records all auditable activity in the file’s audit trail. For more information, see
Audit Trails on page 281.

User Guide | 43

Chapter 4 Using CFX Maestro Dx Software, Security Edition

Signing Secure Files
After saving a file in CFX Maestro Dx SE, users can add an electronic signature. To sign a file, the user’srole must have the permission to save a file. For example, by default the Guest role does not have thepermission to save a file and therefore users assigned this role cannot sign a file.
In CFX Maestro Dx SE, signed files are not set to read-only. They can be reviewed, modified, and signed
multiple times. All changes and signatures are tracked in the file’s audit trail. You can sign the following filetypes:n

Data files (.pcrd)nGene study files (.mgxd)

Note: Files must be saved before they can be signed. If you have recently performed a run in
CFX Maestro Dx SE, save the resulting data file first.

To sign a file1.

Log in to CFX Maestro Dx SE with your Windows login credentials.

2.

Open the secure data file or gene study file to sign.

3.

Choose File > Sign. The Electronic Signature dialog box appears.

44 | CFX Maestro Dx SE Software

4.

Enter your Windows user name and password and the reason for signing the file.
The user name and reason for signing are included in the audit trail (for more information see Audit
Trails on page 281).

5.

Click OK to submit the signature and close the dialog box.

Modifying Secure Files
In CFX Maestro Dx SE, users can modify secure files, including signed and unsigned data and gene study
files. The software prompts you to provide a reason for the change when you save a modified secure dataor gene study file. The changes are tracked in the file’s audit trail.
Tip: Because the software does not create audit trails for plate or protocol files, you are not prompted
to provide a reason when you save changes to these files.

To save a modified data or gene study file1.

Log in to CFX Maestro Dx SE with your Windows login credentials.

2.

Open and modify a secure data file or gene study file.
Tip: For a list of auditable activities, see Auditable Events on page 283.

3.

Choose File > Save. The Audit Trail Change Reason dialog box appears.

User Guide | 45

Chapter 4 Using CFX Maestro Dx Software, Security Edition

This dialog box displays the following information, which is captured in the file’s audit trail header foreach modification event:nDate — the date on which the change occurred.

nUser — the Windows domain and user name of the logged in user.

nComment — the last saved comment.

nSignature — the electronic signature of the last person who signed the file.

nSignature reason — the reason for the signature.

nApplication — CFX Maestro Dx SE (appears as BioRadCFXManager.exe, which is correct).

nApplication version — the current version of CFX Maestro Dx SE.

nFull user — the full name of the logged in user.
Note: This name appears in the audit trail.

nMachine — the computer on which is installed.

The change table displays the auditable changes that occurred as a result of the modification. A brief
description for the reason of the change might also appear.
Tip: You can add or edit descriptions in the Reason for Change column.
4.

Review the list of changes. Provide detailed reasons if necessary.

5.

Do one of the following:n

Click Save Changes to save the changes to the file as well as any changes you made to the table
and close the dialog box.
The changes to the file and the reasons for the changes appear in the file’s audit trail.

n

Click Cancel Changes to revert the file to its previous state and close the dialog box.
The changes are not saved to the file and the audit trail is not updated.

46 | CFX Maestro Dx SE Software

Password Protecting Files
As an added level of security, CFX Maestro Dx SE enables users to set passwords on all secure files.
When setting passwords on a secure file, consider the following conditions:

Condition

ActionNo password is required.

All users can open, modify, and save the secure file,
based on their permissions.

File requires the Save password.

All users can open the secure file, and users who know
the Save password can modify and save the secure file.

File requires the Open password.

Only users who know the Open password can open,
modify, and save the secure file.

File requires both the Open andSome users can open the secure file, and a subgroup of

Save passwords.

those users can modify and save the file.

Depending on the user’s role, any user can perform Save As to create a new secure file with anothername or save a file with the same name in another location as long as one of the following is true:n

The secure file is not password protected.

n

The user has the password to open the file.

Tip: The new file is saved without password protection. The original file retains its passwords.
Depending on the role, a user can modify and save the original file as long as one of the following is true:n

The file is not password protected.

n

The user has the password to open and the password to save the file.

Note: The user’s role must include the right to save files in order to set passwords. For example,
users with the role Guest cannot save files and therefore cannot set passwords on a file.
Important: Only CFX Maestro Dx SE administrators can reset or remove passwords.

User Guide | 47

Chapter 4 Using CFX Maestro Dx Software, Security Edition

To password protect a file1.

Log in to CFX Maestro Dx SE with your Windows credentials.

2.

Open the secure file.

3.

Choose File > File Passwords. The File Passwords dialog box appears.

4.

Enter passwords in the Save Password and Open Password boxes.
Tip: By default, passwords appear as asterisk characters when typed. Select Show Password to
display the password as you type it.
Important: Passwords are case sensitive. CFX Maestro Dx SE does not set limitations on
passwords. For best practice, see your system administrator for password requirements at your
site.

5.

Click Set Passwords to set the passwords and close the dialog box.

6.

Choose File > Save to save the changes to the file.

48 | CFX Maestro Dx SE Software

To remove passwords
Important: You must be a CFX Maestro Dx SE administrator to remove passwords.
1.

In the Password Required dialog box, click Remove Passwords.

The CFX Maestro Dx SE Login dialog box appears.

2.

Provide the Windows user name and password for the CFX Maestro Dx SE administrator and click
OK.
The original data file appears.
Important: You must save the file in order to remove the passwords.

3.

Choose File > Save to save the changes to the file.

User Guide | 49

Chapter 4 Using CFX Maestro Dx Software, Security Edition

To change passwords
Important: Only CFX Maestro Dx SE administrators can change passwords.
1.

Open the secure file.

2.

Choose File > File Passwords. The File Passwords dialog box appears.

Tip: Save Password, Open Password, and Show Password are disabled.
3.

Click Reset Passwords.
The CFX Maestro Dx SE Login dialog box appears.

50 | CFX Maestro Dx SE Software

4.

Provide the Windows user name and password for the CFX Maestro Dx SE administrator and click
OK.
The File Passwords dialog box appears.

5.

6.

Do one of the following:n

To reset password protection, type a new password in the appropriate password box.

n

To remove password protection, clear the password boxes.

Click Set Passwords to save the password changes and exit the dialog box.

User Guide | 51

Chapter 4 Using CFX Maestro Dx Software, Security Edition

52 | CFX Maestro Dx SE SoftwareChapter 5 The Workspace
CFX Maestro Dx Software, Security Edition provides an interface for setting up plates, developing PCR
protocols, running them on CFX Opus Dx Deepwell Dx instruments, and analyzing data from PCR runs.
CFX Maestro Dx SE presents five primary workspaces:n

The Home windown

The Startup Wizardn

The Protocol Editor windown

The Plate Editor windown

The Data Analysis window

Each workspace is shown and briefly described in this chapter.

User Guide | 53

Chapter 5 The Workspace

The Home Window
CFX Maestro Dx SE opens to the Home window and displays the Startup Wizard, from which you can set
up an experiment, perform or repeat a run, or analyze an existing run. From the Home window you canalso view application and instrument logs, create and manage users, and access multiple useful tools. For
more information, see Chapter 6, The Home Window.

54 | CFX Maestro Dx SE Software

The Startup Wizard

The Startup Wizard
Use the Startup Wizard to quickly set up and run user-defined experiments or select and run a PrimePCR
experiment. You can also use this wizard to repeat a run or analyze run data.

User Guide | 55

Chapter 5 The Workspace

The Protocol Editor Window
In the Protocol Editor you can create, open, review, and edit a protocol. You can also modify the lidtemperature for the open protocol. Protocol Editor functionality is detailed in Chapter 7, Creating
Protocols.

56 | CFX Maestro Dx SE Software

The Plate Editor Window

The Plate Editor Window
In the Plate Editor you can create, open, review, and edit a plate. Plate Editor functionality is detailed in
Chapter 8, Preparing Plates.

User Guide | 57

Chapter 5 The Workspace

The Data Analysis Window
In the Data Analysis window you can view and compare run data, perform statistical analyses, export
data, and create publication-ready reports. Data Analysis functionality is detailed in Chapter 10, Data
Analysis Overview and Chapter 11, Data Analysis Details.

58 | CFX Maestro Dx SE SoftwareChapter 6 The Home Window
CFX Maestro Dx Software, Security Edition provides an interface for developing PCR protocols, runningthem on CFX Dx systems, and analyzing PCR run data.
This chapter introduces the CFX Maestro Dx SE and explains the features accessible from the Home
window.

User Guide | 59

Chapter 6 The Home Window

The Home Window
CFX Maestro Dx SE opens to the Home window and displays the Startup Wizard, from which you can set
up a run, perform or repeat a run, or analyze an existing run. From the Home window you can also view
application and instrument logs, create and manage users, and access multiple useful tools.

LEGEND1.
2.

The software title bar displays the name of the software and the logged in user.
The menu bar provides quick access to File, View, Users, Run, Tools, Window, and Help menu
commands.

3.
4.

Toolbar commands provide quick access to menu options.
The left pane displays the instruments connected to the CFX Maestro Dx SE computer andprovides buttons from which you can operate the lid and view the status of the instruments.

5.

The main pane displays the working window. The default working window on the Home screen
is the Startup Wizard.

6.

The status bar displays names of the connected instruments and the logged in user.

60 | CFX Maestro Dx SE Software

The Home Window

File Menu Commands
New — opens a dialog box from which you can choose to create a new protocol, plate, or gene study.
Open — opens a dialog box from which you can choose to navigate to and open an existing protocol,
plate, data file, gene study, LIMS file, run from a stand-alone instrument (stand-alone run), or PrimePCR
run file.
Recent Data Files — displays a list of recently opened PCR files.
Repeat a Run — opens Windows Explorer to the location of saved PCR files, in which you can locate arun to repeat.
Exit — closes CFX Maestro Dx SE.

View Menu Commands
Application Log — displays a software usage log from initial installation to the current day.
Run Reports — displays a list of run reports.
Startup Wizard — displays the Startup Wizard in the main pane.
Run Setup — displays the Run Setup window in the main pane.
Instrument Summary — displays the Instrument Summary window in the main pane.
Detected Instruments — toggles between displaying and not displaying connected instruments in theleft pane. By default, the software displays connected instruments in the left pane.
Toolbar — toggles between displaying and not displaying the toolbar on the top of the screen. By default,
the software displays the toolbar.
Status Bar — toggles between displaying and not displaying the status bar on the bottom of the screen.
By default, the software displays the status bar.
Show — opens a dialog box from which you cannView or block the Status log.

nOpen and view the CFX Maestro Dx SE data folder.

nOpen and view the user’s data folder.

nOpen and view the LIMS file folder.

nOpen and view the PrimePCR folder.

nView the run history.

nView the properties of all connected instruments.

User Guide | 61

Chapter 6 The Home Window

User Menu Commands
Select User — opens the Login screen on which you can select a user from the User Name dropdown list
and log in to the application.
Change Password — opens the Change Password dialog box, in which users can change theirpassword.
Note: This option is disabled for CFX Maestro Dx SE. Users must change their Windows password inorder to change their CFX Maestro Dx SE password.
User Preferences — opens the User Preferences dialog box, in which users can change the default
settings forn

Sending and receiving email notification upon run completionn

Saving data filesnCreating protocols via the Protocol Editor or the Protocol AutoWriternCreating platesnAnalyzing datan

Performing gene expression analysisn

Determining the quality of the datan

Exporting CFX instrument data

User Administration — opens the User Administration dialog box, in which administrators can create
users, modify role permissions, and assign roles to users.
Bio-Rad Service Login — for Bio-Rad technical service staff use only. Do not select this command.

Run Menu Commands
User-defined Run — opens the Run Setup window, in which you can set up a user-defined protocol andplate, and then run a PCR experiment on the selected instrument.
PrimePCR Run — opens the Start Run tab in the Run Setup window with the default PrimePCR protocol
and plate layout loaded based on the selected instrument.
End-Point Only Run — opens the Start Run tab in the Run Setup window with the default end-point
protocol and plate layout loaded based on the selected instrument.
Qualification Run — opens the Start Run tab in the Run Setup window with the default Bio-Rad
qualification protocol and plate layout loaded for the selected instrument.

62 | CFX Maestro Dx SE Software

The Home Window

Tools Menu Commands
Master Mix Calculator — opens the Master Mix Calculator, in which you can create a reaction mixture
and print the calculations.
Protocol AutoWriter — opens the Protocol AutoWriter dialog box, in which you can easily create a newprotocol.
Ta Calculator — opens the Ta Calculator, in which you can easily calculate the annealing temperature ofprimers.
Dye Calibration Wizard — opens the Dye Calibration wizard, in which you can calibrate an instrument
for a new fluorophore.
Reinstall Instrument Drivers — reinstalls the drivers that control communication with Bio-Rad’s
real-time PCR systems.
Zip Data and Log Files — opens a dialog box in which you can select files to condense and save in a
zipped file for storage or to email.
Batch Analysis — opens the Batch Analysis dialog box, in which you can set parameters for analyzing
more than one data file at a time.
Options — opens a dialog box in which you cannConfigure your email server settingsnConfigure export settings for LIMS, Seegene, and other data files
Tip: You can also select the option to automatically start the Seegene Viewer upon export if
you choose to export your data in Seegene format.

nChange the language that the user interface displays (English, Chinese, Russian)
Important: You must restart CFX Maestro Dx SE to display the selected language.
Important: Your operating system's language must correspond to the language you want todisplay in the CFX Maestro Dx SE interface.

Help Menu Commands
Tip: The Help menu is available on the menu bar in all CFX Maestro Dx SE windows.
Contents — displays the Contents tab in the CFX Maestro Dx SE Help system.
Index — displays the Index tab in the CFX Maestro Dx SE Help system.
Search — displays the Search tab in the CFX Maestro Dx SE Help system.
Open User Guide — opens a PDF of this guide.

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Chapter 6 The Home Window

Additional Documentation — provides access to the CFX Opus Dx Real-Time PCR Systems Operation
Manual.
Release Notes — opens the Release Notes document for the installed version of CFX Maestro Dx SE.
Video Resources — opens a website where Bio-Rad video resources, such as instructional videos,reside.
qPCR Applications and Technologies Web Site — opens Bio-Rad’s qPCR Applications &Technologies web site, from which you can learn more about real-time PCR (qPCR).
PCR Reagents Web Site — opens Bio-Rad’s PCR and qPCR reagents website, from which you canorder PCR reagents, supermixes, dyes, and kits.
PCR Plastic Consumables Web Site — opens Bio-Rad’s PCR Plastics and Consumables website, fromwhich you can order PCR plates, plate seals, tubes and caps, and other plastics accessories.
Software Web Site — opens Bio-Rad’s PCR Analysis Software website, from which you can order
updated versions of Bio-Rad’s CFX Maestro Dx SE.
About — displays CFX Maestro Dx SE copyright and version information.

Toolbar Commands— opens Windows Explorer, in which you can navigate to and open a data file or a gene study file.

— opens the Master Mix Calculator.

— opens the Run Setup window.

— opens the Run Setup window with the default PrimePCR protocol and plate layout loaded basedon the selected instrument.

— opens the Startup Wizard.

64 | CFX Maestro Dx SE Software

The Home Window

The Startup Wizard
When CFX Maestro Dx SE starts, the working pane displays the Startup Wizard. From the Startup Wizard
you cannSelect an instrument from the detected instruments and set up a user-defined or PrimePCR runnOpen and repeat a runnOpen a data file to analyze results from a single run or a gene study file for results from multiple gene
expression runs

These tasks are explained in detail in the chapters that follow.

Status Bar
The left side of the status bar at the bottom of the main software window displays the current status of thedetected instruments. The right side of the status bar displays name of the current user and the date andtime.

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Chapter 6 The Home Window

Detected Instruments PaneThe Detected Instruments pane displays each instrument that is connected to the CFX Maestro Dx SE
computer. By default, each instrument appears as an icon, and its serial number appears as its name.
From this pane you cannView the properties and calibrated dyes for the selected instrument
For information about instrument properties, see Viewing the Properties of an Instrument on page 69.

nView the status of a connected instrumentnOpen the motorized lid on the selected instrumentn

Close the motorized lid on the selected instrumentnView the status of all connected instruments

To view the status of a connected instrumentuIn the Detected Instruments pane, select the target instrument and do one of the following:n

Click View Status in the Selected Instrument section.

nRight-click and select View Status on the menu that appears.

The Run Details dialog box appears displaying the Run Status tab. The status of the selected
instrument appears below the run status pane, for example:

To open or close the lid of an instrumentuIn the Detected Instruments pane, select target instrument and do one of the following:n

Click Open Lid or Close Lid in the Selected Instrument section.

66 | CFX Maestro Dx SE Software

The Home Windown

Right-click and select the appropriate action on the menu that appears.

nOpen the Run Details dialog box, select Run Status tab, and click Open Lid or Close Lid.

To view the status of all detected instrumentsu

Do one of the following:n

In the All Instruments section in the Detected Instruments pane, click View Summary.

n

On the menu bar, select View > Instrument Summary.

The Instrument Summary dialog box appears.
Tip: If the system detects only one connected instrument, the All Instruments section does notappear in the Detected Instruments pane. To view the instrument summary for a single
instrument, select View > Instrument Summary.

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Chapter 6 The Home Window

Instrument Summary Toolbar Controls
Table 5 lists the controls and functions in the Instrument Summary toolbar.
Table 5. Instrument Summary Toolbar Controls

ButtonButton Name

Function

Create a new Run

Creates a run on the selected block by
opening the Run Setup window.

Stop

Stops the current run on selected blocks.

Pause

Pauses the current run on selected
blocks.

ResumeResumes the run on selected blocks.

Flash Block Indicator

Flashes the indicator LED on the lid of theselected blocks.

Open Lid

Opens the selected block’s motorized lid.

Close Lid

Closes the selected block’s motorized lid.

Hide Selected Blocks

Hides the selected blocks in theInstrument Summary list

Show All Blocks

Displays the selected blocks in theInstrument Summary list

Show

Select which blocks to show in the list.
Select one of the options to show all
detected blocks, all idle blocks, all blocks
that are running with the current user, or
all running blocks

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Viewing the Properties of an Instrument
From the Detected Instruments pane you can view details about a selected instrument, including its
properties and a list of its calibrated dyes (fluorophores).

To view instrument propertiesu

In the Detected Instruments pane, right-click the target instrument and select Properties on the menu
that appears.

Properties Tab
The Properties tab lists technical details about the selected instrument including the model, serial
numbers of its components, and firmware versions. The default name of the instrument (its serial number)appears in many locations, including the Detected Instruments pane and in the header bar of theInstrument Properties dialog box. You can rename the instrument in order to more easily identify it.
Note: You cannot change the name of the CFX Opus instrument using CFX Maestro.

Calibrated Dyes Tab
The Calibrated Dyes tab displays the calibrated fluorophores and plates for the selected instrument.

To see detailed information about a calibration, click its Info button in the Detail column.

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Chapter 6 The Home Window

Before You Begin
This section explains tasks you might need to perform before using CFX Maestro Dx SE. This includesnCreating a reaction master mixnCalibrating new dyes

Creating a Reaction Master Mix
Using CFX Maestro Dx SE’s Master Mix Calculator, you can easily calculate the required volume of eachcomponent in your master mix. You can print the master mix calculation table to your default printer, andsave the calculations for each target for later use.

To create a reaction master mix using the Master Mix Calculator
1.

To open the Master Mix Calculator, do one of the following:nSelect Tools > Master Mix Calculator.

n

Click Master Mix Calculator on the toolbar.

The Master Mix Calculator appears.

70 | CFX Maestro Dx SE SoftwareBefore You Begin

2.

In the Reaction section, select a detection method:n

SYBR® Green/EvaGreen®

nProbes3.

To create a new target, in the Target section click Create New. A new target name appears in thetarget dropdown list.

4.

(Optional) To change the default target name:a.

Highlight the target’s name in the dropdown target list.

b.

Type a new target name in the Target box.

c.

Press the Enter key.

5.

Adjust the starting and final concentrations for the forward and reverse primers and any probes.

6.

In the Master Mix Setup section, adjust the values fornNumber of reactions to run

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Chapter 6 The Home Windown

Reaction volume per wellnTemplate volume per wellnSupermix concentration per welln

Excess reaction volume per well

7.

(Optional) Perform steps 2–6 for as many targets as necessary.

8.

In the Choose Target to Calculate section, select the target to calculate.
Tip: You can calculate only one or several or all targets at the same time.
The calculated volumes of the required components for each selected target appear in the mastermix table.

9.

Click Set as Default to set the quantities input in the Target and Master Mix Setup sections as newdefaults.

10. Click OK to save the contents of the Master Mix Calculator dialog box.

To print the master mix calculations tableu

To print a master mix calculations table, click Print.
The calculations table prints to your default printer.

To save the master mix calculations table as a PDF
uChange your default printer to a PDF driver and click Print on the Master Mix Calculator.

To delete targetsu

Select the target using the dropdown target list and click Remove.
Important: Removing a target from the target list also removes it from any master mix
calculations it is used in. Take care when deleting a target.

Calibrating New Dyes
The CFX Opus 96 Dx and CFX Opus Deepwell Dx systems are factory calibrated for commonly usedfluorophores in white-well and clear-well plates. The CFX Opus 384 Dx systems are factory calibrated forcommonly used fluorophores in white-well plates only. Table 6 lists the fluorophores and channel for which
each instrument is calibrated.
Note: The CFX Opus 96 Dx, CFX Opus 384 Dx and CFX Opus Deepwell Dx systems also include achannel dedicated to FRET chemistry. This channel does not require calibration for specific dyes.

72 | CFX Maestro Dx SE SoftwareBefore You Begin

Important: If you conduct a user-defined calibration of a dye that was factory calibrated, theinstrument uses the user-defined calibration instead of the factory calibration.
Table 6. Factory calibrated fluorophores, channels, and instruments

Fluorophores

ChannelExcitation, nm

Detection, nm

Instrument

FAM, SYBR®

1

450–490515–530

CFX Opus 96

Green IDx, CFX Opus
384 Dx, and
CFX Opus
Deepwell Dxsystems

VIC, HEX, CAL

2

515–535

560–580

CFX Opus 96 Dx

Fluor Gold 540, Cal,CFX Opus 384

Fluor Orange 560

Dx, and
CFX Opus
Deepwell Dxsystems

ROX, Texas Red,3

560–590

610–650

CFX Opus 96

CAL Fluor Red 610,

Dx, CFX Opus

TEX 615384 Dx, and
CFX Opus
Deepwell Dxsystems

Cy5, Quasar 670

4

620–650

675–690

CFX Opus 96
Dx, CFX Opus
384 Dx, and
CFX Opus
Deepwell Dxsystems

Quasar 705, Cy5.5

5

672–684705–730

CFX Opus 96 Dx
systems only

FRET Chemistry (Not Factory-Calibrated)
Non-factory
calibrated color

FRET

450–490560-580

CFX Opus 96
Dx, CFX Opus
384 Dx, and
CFX Opus
Deepwell Dxsystems

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Chapter 6 The Home Window

To calibrate new dyes for CFX systems
1.

In the Home window, select a target instrument in the Detected Instruments pane.

2.

Select Tools > Calibration Wizard to open the Dye Calibration wizard.

Fluorophores already calibrated for the target instrument appear in the Calibrated Fluorophores
table.
3.

In the Calibrate New or Existing Fluorophores section, select the fluorophore to calibrate from thedropdown list.
If the fluorophore name is not included in the list, type its name in the text box to add it to the list.
Important: Take care when naming custom calibrated fluorophores. If you create a custom dye
calibration for a fluorophore with the same name as a factory calibrated fluorophore, the custom
fluorophore (not the factory calibrated fluorophore) will be the one used by the instrument during
the runs.

4.

Select the plate type for the fluorophore.
If the plate type is not included in the list, type the name in the text box to add it to the list.

5.

Select a channel for the fluorophore.

6.

Select a plate column for the fluorophore.

74 | CFX Maestro Dx SE SoftwareBefore You Begin

7.

(Optional) Type a color to associate with the fluorophore.

8.

Click Add to Plate to add the fluorophore.

9.

(Optional) Repeat steps 3–8 to add each fluorophore you plan to calibrate for the plate.

10. When you finish adding fluorophores, click View Plate to open the Pure Dye Plate Display window.
Use this window as a guide for loading dyes into the plate.
11. Prepare a 96-, 384-, or deep well plate for dye calibration:a.

Pipette dye solution into each well, following the pattern shown in the Pure Dye Plate Display.

b.

For each fluorophore, fill four wells with 50 µl (96-well or deep well plate), 30 µl (384-well plate) of
300 nM dye solution. Notice that at least half the plate contains blank wells.

c.

Seal the plate using the sealing method you will use in your experiment.

12. Place the calibration plate in the block and close the lid.
13. In the Dye Calibration wizard, click Calibrate and then OK to confirm that the plate is in the block.
14. When CFX Maestro Dx Software, Security Edition completes the calibration run, a dialog box
appears. Click Yes to finish calibration and open the Dye Calibration Viewer.
15. Click OK to close the window.

Setting User Preferences
Tip: It is not required to perform these tasks in order to use CFX Maestro Dx SE. You can safely skip
this section or perform these tasks at any time.
In CFX Maestro Dx SE, each user can customize his or her working environment. For example, in theUsers > User Preferences menu, you can do the following:n

Set up email notification of run completion.
Note: This feature is available only to users whose role has been given this right. See Managing
CFX Maestro Dx Software, Security Edition User Roles on page 41 for more information.

nChange the default settings foro

The location in which to save filesoThe run setup filesoThe file naming prefixn

Set the default parameters to use when creating a new protocol and plate.

n

Set the default data analysis and gene expression parameters.

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Chapter 6 The Home WindownCustomize the default quality control parameters.

nCustomize data export parameters.

In the Tools menu, you can do the following:nCreate a master mix.

n

Calibrate dyes for a specific instrument.

Note: The master mix and dye calibration are available to anyone who logs in to the software.
This section explains how to perform these tasks.

Setting Up Email Notification
You can connect CFX Maestro Dx SE to your outgoing email server to send email notification of run
completion to a list of users. You can also choose to attach a data file and an analysis report to the list ofusers. To set up the connection between CFX Maestro Dx SE and your SMTP server, see Connecting
Security Edition to an SMTP Server on page 77.
Note: A user’s ability to access email setup features depends on the user’s role and permissions
assigned by the administrator. For details on managing users and their roles, see Managing
CFX Maestro Dx Software, Security Edition User Roles on page 41.

To set up email notifications
1.

Select User > User Preferences to open the User Preferences dialog box.
The User Preferences dialog appears displaying the Email tab.

Note: You are informed if the system detects that you have not set up a valid SMTP server for
CFX Maestro Dx SE. Click Configure Outgoing Email to open the Options dialog box andconfigure the email SMTP server. For more information, see Connecting Security Edition to an
SMTP Server on page 77.

76 | CFX Maestro Dx SE SoftwareBefore You Begin

2.

In the To text box, type the email address of each person who you plan to inform of run completion.
All recipients will receive email after the run completes.
Note: You must enter each email address on a separate line. Press Enter or Return after eachaddress.

3.

(Optional) In the cc text box, type the email address of any recipient to whom you plan to send a copyof each email notification.

4.

(Optional) By default, all recipients receive a copy of the data file as an attachment. Clear thischeckbox if you do not want to attach a copy of the data file.

5.

(Optional) Select Attach Analysis Report to attach a PDF of the analysis report to the email.

6.

Click OK to save the changes and close the User Preferences dialog box.

Note: You might be able to configure the system to send an email notification to your cell phone,depending on your service provider. Contact your cell phone service provider for specific information
regarding the email address for your cell phone. Enter your phone's email address (for example,
5552221234@your_service_provider_EmailDomain.net) in the To text box of the User Preferences
screen.

To edit a recipient’s email addressuModify the email address as necessary and click OK.

To remove an email recipient
1.

Select the email recipient and press the Delete key.

2.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

Connecting Security Edition to an SMTP Server
Important: Some commercial webmail service providers have increased email security. If you use
these accounts, you must enable the setting Allow less secure apps in their account settings toenable CFX Maestro Dx SE to send email. See the security information for your webmail service
provider for more information.
If you are using Google Gmail or Microsoft Office 365 SMTP server to send email, you must enable a 2factor verification and generate an "App Password" in your Gmail or Office365 account settings. For
authentication in the Maestro Email Setup dialog, copy and paste the “App Password” into the Password
field instead of regular email password.

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Chapter 6 The Home Window

You must establish a connection from CFX Maestro Dx SE to your email server before the software cansend email notifications.

To connect CFX Maestro Dx SE to an email server
1.

Do one of the following:nSelect User > User Preferences and click Configure Outgoing Email on the Email tab.

n

Select Tools > Options.

The Options dialog box appears displaying the Email tab.

2.

Provide the following information for your company:n

SMTP Server Name — the name of the outgoing email server at your company.

nPort — the port number of your SMTP server. This is usually 25.

n

Use SSL — Secure Sockets Layer (SSL) option. Some SMTP servers require this setting. If it is
not required at your company, clear this checkbox.

n

Use Default “From” Address — the name of the email server at your company. Some SMTP
servers require all sent email to have a “from” address that is from a certain domain, for example,
name@YourCompany.com. If that is the case, clear this checkbox and provide a valid email
address.

nAuthentication Required — if your site requires account authentication, verify that thischeckbox is selected.

nUser Name — the name of the authenticated account. This is required only if Authentication
Required is selected.

78 | CFX Maestro Dx SE SoftwareBefore You Beginn

Password — the password for the authenticated account. This is required only if Authentication
Required is selected.

Important: If you are using Google Gmail or Microsoft Office 365 SMTP server to send email,you must enable a 2-factor verification and then generate an “App Password” in your Gmail orOffice365 account settings. For authentication in Maestro Email Setup dialog, copy and paste
the “App Password” into the CFX Maestro Dx SE's Password field instead of regular email
password.
To verify that the SMTP server settings are correct, enter a valid email address in the Test Email
Address text box and click Test Email.
Note: Some SMTP servers do not allow attachments and others allow attachments only up to a
specific size. If you plan to email data files and/or reports using CFX Maestro Dx SE, select Test
Attachment and set Attachment Size in MB to 5 megabytes (MB) or more.
3.

Click OK to save the changes and close the dialog box.

Changing the Default File Settings
In the Files tab on the User Preference dialog box, you can changenThe default location in which to save CFX Maestro Dx SE filesn

The default files for run setupn

The default file naming parameters

To change the default file settings
1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Files tab.

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Chapter 6 The Home Window

3.

In the Default Folder for File Creation section, navigate to and select a default folder in which you
want to save new files. You can select a different location for each file type:n

Protocol

n

Plate

n

Data Filen

Gene Study

4.

In the File Selection for Run Setup section, navigate to and select the target protocol and plate files toappear when you open the Experiment Setup window.

5.

In the Data File section, define the prefix and/or suffix for data files. For any part, select a new value
from its dropdown list. You can also provide custom prefix and suffix values in the Prefix and Suffix
text boxes.
CFX Maestro Dx SE displays a preview of the file name below the selection boxes.

6.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

80 | CFX Maestro Dx SE SoftwareBefore You Begin

Setting the Default Protocol Parameters
To set default protocol parameters for the Protocol Editor and Protocol AutoWriter1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Protocol tab.

3.

In the Protocol Editor section, specify values for the following settings that appear in the Protocol
Editor:4.

5.

nSample volume — the volume of each sample in the wells (in µl).

n

Lid Shutoff temperature — the temperature in ºC at which the lid heater turns off during a run.

In the Protocol AutoWriter section, specify values for the following settings that appear in the Protocol
AutoWriter:nAnnealing temperature — the temperature in ºC for experiments that use iProof DNApolymerase, iTaq DNA polymerase, or other polymerases.

n

Amplicon length — the length of the amplicon in bp.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

Setting Default Plate Parameters
Changes that you make to the Plate tab are available to all users of the software. Changes that you makeduring plate setup are available to users after you save and close the plate file.
In the User Preferences dialog box you can do the following:

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Chapter 6 The Home Windown

Set default plate parameters.

n

Add new target, sample, and biological group names to their respective libraries.

n

Delete target, sample, and biological group names from their respective libraries.

To set the default plate parameters
1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Plate tab.

3.

Specify values for the following settings for a new plate file. These values appear in the Plate Editor
window:n

Plate typen

Plate sizenUnits — the concentration of the starting template for wells that contain standards.
CFX Maestro Dx SE uses these units to create a standard curve in the Data Analysis
Quantification tab.

n

Scientific notation — when selected, CFX Maestro Dx SE displays the concentration units inscientific notation.

n

Scan mode — the number or type of channels to scan during a run.

82 | CFX Maestro Dx SE SoftwareBefore You Begin

4.

nFluorophores — the default fluorophores that appear in the Plate Editor well loading controls.

nLibraries — the target, sample, and biological group names that you typically use in yourexperiments:oTarget names — the names of target genes and sequences.

oSample names — the names of experiment samples or an identifying characteristic for the
samples (for example, Mouse1, Mouse2, Mouse3).

oBiological group names — the names for groups of similar samples that have the same
treatment status or conditions (for example, 0Hr, 1Hr, 2Hr).

Click OK to save the changes and close the dialog box.

To add a new target, sample, or biological group nameu

In the appropriate library box, type the name for the target, sample, or biological group and click OK.

To delete a target, sample, or biological group nameu

In the appropriate library box, select the name and press the Delete key and then click OK.

Important: Names that you remove from the library are removed from the software and are no longer
available to users. To restore the default CFX Maestro Dx SE names, click Restore Defaults. Clicking
Restore Defaults in the User Preferences dialog box resets all preferences on all tabs to the original
factory settings. Take care when deleting default CFX Maestro Dx SE names and when clicking thisbutton.

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Chapter 6 The Home Window

Setting Default Data Analysis Parameters
To set default Data Analysis parameters
1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Data Analysis tab.

3.

In the Analysis Mode section, select the mode in which to analyze the data (either Fluorophore or
Target).

4.

In the PCR Quantitation section, set default parameters for the following options:nBaseline Setting — the baseline method for analysis mode.

n

Cq Determination Mode — the mode in which Cq values are calculated for each fluorescencetrace (either regression or single threshold).

nThreshold Calc. Mode — the end-point target amount.
The default is Auto. That is, the software automatically calculates the end-point target. To set a
specific threshold, clear the Auto checkbox and enter your end-point amount, calculated inrelative fluorescence units (or RFU). The maximum value is 65000.00 RFUs. Data files for
subsequent runs will use this threshold setting.

84 | CFX Maestro Dx SE SoftwareBefore You BeginnBaseline Calc. Mode — the baseline value for all traces.
The default is Auto. That is, the software automatically calculates the baseline for all traces. To
set a specific baseline value, clear the Auto checkbox and enter minimum and maximum values
for the cycle range (1 to 9999). Data files for subsequent runs will use this cycle range.

n5.

Log View — determines how the software displays the amplification data:oOn — the amplification data are displayed in a semilogarithmic graph.

o

Off — (the default) the amplification data are displayed in a linear graph.

In the End Point section, select the number of end cycles to average when calculating the end-point
calculations:n

PCR — the number of end cycles to average for quantification data (default is 5).

n

End Point Only run — the number of end cycles to average for end-point data (default is 2).

6.

In the Melt Curve section, select the peak type to detect (either positive or negative).

7.

In the Well Selector section, select how to display well labels (by sample type, target name, or samplename).

8.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

Setting Default Gene Expression Data File Parameters
To set the default parameters for a new gene expression data file1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Gene Expression tab.

3.

Specify the values for the following settings:nRelative to — graphs the gene expression data relative either to a control (originating at 1) or tozero:oZero — the software ignores the control. This is the default when no control sample isassigned in the Experiment Settings window.

oControl — the software calculates the data relative to the control sample assigned in theExperiment Setup window.

n

X-axis — graphs the sample or the target on the x-axis.

nY-axis — graphs linear, log2, or log10 scale on the y-axis.

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Chapter 6 The Home Windown

Scaling — the scaling option for the graph (the default option is unscaled):oHighest — the software scales the graph to the highest data point.

oLowest — the software scales the graph to the lowest data point.

oUnscaled — the software presents the data unscaled in the graph.

nMode — the analysis mode, either relative quantity (∆Cq) or normalized expression (∆∆Cq).

nError Bar — the data variability presented as either the standard deviation (Std. Dev.) or the
standard error of the mean (Std. Error Mean).

nError Bar Multiplier — the standard deviation multiplier used to graph the error bars
(default is 1).
You can increase the multiplier to either 2 or 3.

nSample Types to Exclude — the sample types to exclude from the analysis.
You can select one or more sample to exclude from the analysis. To exclude all sample types,
clear the checkboxes of any selected sample types.

4.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

Customizing Quality Control Rules
In CFX Maestro Dx SE, you can set quality control rules, which are applied to data in the Data Analysis
window. The software validates the data against the rules that you set.
Note: By default, all quality control rules are enabled.
Tip: You can easily exclude wells that fail a QC parameter from analysis in the QC module of theData Analysis window.

To customize quality control rules
1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the QC tab.

86 | CFX Maestro Dx SE SoftwareBefore You Begin

Where:

3.

4.

n

NTC — no template controln

NRT — no reverse transcriptase control

nEfficiency — reaction efficiencyn

Std Curve R^2 — R square value for the standard curvenReplicate group Cq Std Dev — standard deviation calculated for each replicate group

For each QC rule, do one of the following:n

To use its default value, do nothing.

n

To change its value, click its Value text box, type a new value, and press the Enter key.

n

To disable the rule, clear its Use checkbox.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

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Chapter 6 The Home Window

Customizing Data Export Parameters
You can export CFX Maestro Dx SE data in the following formats:n

Text (.txt)n

CSV (.csv)

nExcel (.xls, .xlsx)n

XML (.xml)

nHTML (.html)

Important: Your computer must have Microsoft Excel installed in order for you to export data to a
Microsoft Excel spreadsheet.
You can specify the type of data to export and customize the output of the exported data.

To customize data export parameters
1.

Select User > User Preferences to open the User Preferences dialog box.

2.

In the User Preferences dialog box, select the Custom Export tab.

3.

On the Export Format dropdown list, select a format in which to export the data.

88 | CFX Maestro Dx SE SoftwareBefore You Begin

4.

In the Data to Export section, select or clear the checkboxes for the type of data to export. The
selected items appear in the Exported Columns list box.
Note: By default, the run information is included in the header. Clear this checkbox if you do notwant the run information included.

5.

You can change the output display order of the selected items.
In the Exported Columns list box, highlight the item and then click the arrow buttons to the left of thelist to move it up or down.

6.

Optionally, you can change the output column names of the selected items:a.

Click Customize Column Names.
The Column Name Customizer dialog box appears.

7.

b.

For each default column name that you want to change, type the new name in its Custom Name
field.

c.

Do one of the following:n

Click OK to save the changes and return to the Custom Export tab. The new name appears
in parentheses beside the default column name in the Exported Columns list box.

n

Click Cancel to clear the changes and return to the Custom Export tab.

Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all preferences on alltabs to the original factory settings. Take care when clicking this button.

User Guide | 89

Chapter 6 The Home Window

90 | CFX Maestro Dx SE SoftwareChapter 7 Creating Protocols
A protocol is set of steps that are executed in a specific sequence. In CFX Maestro Dx Software, Security
Edition, all of the steps are associated with options on the instrument. For example, the steps instruct the
instrument to control block and lid temperature, apply a temperature difference across the block, take aplate read, or perform a melt curve analysis. Each option is specified for different plate and run types.
CFX Maestro Dx SE provides two options for creating protocols: Protocol Editor and Protocol AutoWriter.
The Protocol Editor features include the following:nStandard protocol controls to quickly create protocolsnAbility to quickly calculate a gradient for the selected number of rowsnAbility to quickly calculate run time for the selected plate typen

Ability to edit protocol stepsnAbility to save protocols for reusenAbility to print the protocol to a default printer

The Protocol AutoWriter automatically generates a customized PCR protocol with hot start, initialdenaturation, annealing, and extension steps using parameters that you provide. You can then view agraphical representation of the suggested protocol and edit, run, or save the protocol.

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Chapter 7 Creating Protocols

Parameters and Ranges for Protocol StepsUse the information in Table 7 to modify the default settings for the steps in your protocol.

Temperature Steps
The target temperature is a value between 4.0 and 100.0 °C, set in tenths of a degree. The system ramps
up to this temperature and holds that value for a specified amount of time (the hold time).

Gradient Steps
The gradient range is the difference between the lower and upper temperatures in a gradient step. The
maximum allowed range is 24°C. The lower temperature is a value between 30.0 and 99.0°C, set intenths of a degree. The maximum upper temperature is 100°C. The thermal cycler ramps up to the target
temperature gradient across the block and holds that temperature for a specified hold time.
Important: The instrument calculates the gradient value. When you enter a value in the gradientcalculator’s top and bottom fields, the software automatically calculates and assigns thetemperatures for the remaining fields. When you enter a temperature in any field between the top andbottom field, the instrument automatically calculates the remaining fields. You cannot manually entera temperature value in every field.
Table 7. Parameters and ranges for protocol steps

Parameter

Range

Description

Ramp raten For CFX Opus 96 Dx systems:

Instructs the thermal cycler to ramp to the

0.1–5°C per sec
n For CFX Opus 384 Dx systems:
0.1–2.5°C per sec

target temperature at the specified rate inthat step.
Available only to temperature steps.

n For CFX Opus Deepwell Dxsystems:
0.1–2.5°C per sec
Increment

A number from –10.0 to 10.0°C per

Instructs the thermal cycler to change thecycle in tenths of a degree

target temperature of a step with eachcycle, where a positive number increases
the temperature and a negative number
decreases the temperature.
Available only to temperature steps.

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Parameters and Ranges for Protocol Steps

Table 7. Parameters and ranges for protocol steps, continued

Parameter

Range

Description

ExtendA time from –60 to 60 sec per cycle

Instructs the thermal cycler to extend thehold time with each cycle. A positive
number increases the hold time and anegative number decreases the hold time.
Available to both temperature and gradientsteps.

Beep(No parameters)

Instructs the thermal cycler to beep to signalthat the thermal cycler has reached thetarget temperature for that step.
Available only to temperature steps.

Plate read(No parameters)

Instructs the thermal cycler to add a plate
read to the selected step.
Available to both temperature and gradientsteps.

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Chapter 7 Creating Protocols

Protocol Editor Window
Use the Protocol Editor to create, open, review, and edit a protocol. By default, the Protocol Editor
displays a generic real-time 2-step protocol for a 96-well plate.

LEGEND
1.
2.

The menu bar provides quick access to the File, Settings, and Tools menu commands.
The toolbar provides quick access to save and print the protocol, determine where to insert astep, set sample volume, and view estimated protocol run time.

3.

The main pane displays a graphical representation of the protocol.

4.

The lower pane displays the protocol outline.

5.

The left pane displays the protocol controls that you can add to customize the protocol.

File Menu Commands
Save — saves the current protocol.
Save As — saves the current protocol with a new name or in a new location.

94 | CFX Maestro Dx SE SoftwareProtocol Editor Window

File Passwords — enables users to set their file save and file open passwords.
Tip: For more information, see Password Protecting Files on page 47.
Close — closes the Protocol Editor.

Settings Menu Command
Lid Settings — opens the Lid Setting dialog box in which you can change or set the lid temperature.

Tools Menu Commands
Gradient Calculator — opens a dialog box from which you can select the block type for a gradient step.
The default is 96 wells.
Run time Calculator — opens a dialog box from which you can select the plate type and scan mode inorder to calculate the estimated run time in the Run Setup window. The default is 96 wells, all channels.

Toolbar Commands— saves the current protocol file.

— prints the selected window.

— use this command to select where to insert steps relative to the currently
selected step.

— use this command to enter a sample volume in µl. Sample volumes differ
depending on the type of block:n

For a 96-well block the range is 0–50 µl.

n

For a 384-well block the range is 0–30 µl.

n

For a 96-deep well block the range is 0–125 µl.

— displays the estimated run time based on the protocol steps, ramp rate, andthe type of block selected.

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Chapter 7 Creating Protocols— displays Help information about protocols.

Protocol Editing Controls
The left pane of the Protocol Editor window comprises controls that you can use to create protocols.
Each control consists of a set of parameters that represent a step in the protocol. You can modify eachparameter and add or remove them to customize your protocol. This section describes the options in eachcontrol.
nInsert Step — inserts a step before or after the selected step. You can
edit the temperature and hold time values either in the protocol’s
graphical display or in the protocol outline.

nInsert Gradient — inserts a gradient step based on the type of well blockselected in the gradient calculator. You can edit the gradient range in theGradient pane that appears when a gradient step is inserted.

nInsert GOTO — inserts a cycling (loop) step, which informs the softwareto repeat specific steps in sequence for a specified number of cycles. The
repetitions begin after the first cycle is complete. For example, you can
inform the software to perform 39 repetitions of steps 2–4. After the final
repetition, the software will have performed steps 2–4 a total of 40 times.
You can edit the return-to (GOTO) step and the number of cycles either in the graphical display or in
the protocol outline.
nInsert Melt Curve — inserts a melt curve read step.

n

Insert Plate Read to Step — adds a plate read command to the selected step. A plate readmeasures the amount of fluorescence at the end of a cycle. The plate read step is generally the last
step in a GOTO loop.
Tip: After you add a plate read command to a step, the button changes to Remove Plate Readwhen you select the step.

nRemove Plate Read — removes a plate read command from the selected step.
Tip: After you remove a plate read command from a step, the button changes to Add Plate Readto Step when you select the step.

n

Step Options — opens the Step Options dialog box and displays the options available for theselected step. See Step Options on page 97 for detailed information about the step options.
Tip: You can also access the Step Options by right-clicking the step in the graphic display.

nDelete Step — deletes the selected step from the protocol.

96 | CFX Maestro Dx SE SoftwareProtocol Editor Window

Step Options
Open the Step Options dialog box to view the options you can add, change, or remove from a step.

n

Plate Read — when selected, adds a plate read to the step.

n

Temperature — sets the target temperature for the selected step.

n

Gradient — sets the gradient range for the step; the range is 1–24°C.
Note: A gradient runs with the lowest temperature in the front of the block (in this image, row H)
and the highest temperature in the back of the block (in this image, row A).

n

Increment — the amount to increase (or decrease) the temperature of the selected step; this value
amount is added to the target temperature with each cycle. The range is ±0.1–10°C.
Note: To decrease the temperature, type a minus sign (–) before the numerical value(for example, –5°C).

nRamp Rate — the ramp rate for the selected step; the range depends on the block size.

nTime — the hold time for the selected step.

nExtend — the amount of time (in sec) to extend or decrease the selected step; this option is added tothe hold time in each cycle; the range is ±1–60 sec.

n

Beep — when selected, a beep sounds during the step.
Tip: When you enter a number that is outside the option range, the software changes thenumber to the closest entry within the range.

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Chapter 7 Creating Protocols

Creating a Protocol in the Protocol Editor
Using the Protocol Editor, you can create custom protocol files. You can also edit and save previously
saved protocol files or sample protocol files shipped with CFX Maestro Dx SE.
To create a new protocol file, perform the following:nOpen a protocol file in the Protocol Editor.
Tip: You can open a new or existing protocol in the Protocol Editor.

n

Set up the new protocol.

n

Add steps to the protocol from the protocol controls pane.

n

Edit the properties of the steps.

n

Save the protocol.

Tip: To create a new protocol from a previously saved or sample protocol file, see Opening an
Existing Protocol in the Protocol Editor on page 99.

Opening a New Protocol File in the Protocol Editor
CFX Maestro Dx SE offers multiple options to open a new protocol file:n

From the File menu in the Home windown

From the Run Setup dialog box in the Home windown

From the Startup Wizard dialog box in the Home window

To open a new protocol file from the File menuu

In the Home window, select File > New > Protocol.
The Protocol Editor window opens, displaying the default protocol file.
Tip: For information about setting your default protocol, see Changing the Default File Settings
on page 79.

To open a new protocol from the Run Setup dialog box
1.

In the Home window, do one of the following to open the Run Setup dialog box:n

Select Run > User-defined Run.

n

Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab and displays your default protocol file.

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Creating a Protocol in the Protocol Editor

2.

Click Create New.
The Protocol Editor window opens, displaying the default real-time protocol.

To open a new protocol file from the Startup Wizard
1.

In the Home window, do one of the following to open the Startup Wizard if it is not in view:nSelect View > Startup Wizard.

n

Click Startup Wizard on the toolbar.

2.

If necessary, select the instrument type from the dropdown list.

3.

Click User-defined as the run type.
The Run Setup dialog box opens to the Protocol tab and displays the default protocol file.

4.

Click Create New.
The Protocol Editor window opens, displaying the default real-time protocol.

To open a new protocol from the Run menu
1.

In the Home window, do one of the following to open the Run Setup dialog box:n

Select Run > User-defined Run.

n

Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab and displays your default protocol file.
2.

Click Create New.
The Protocol Editor window opens, displaying the default real-time protocol.

Opening an Existing Protocol in the Protocol Editor
CFX Maestro Dx SE provides sample protocol files that you can edit and save as custom new protocols.
You can also create a new protocol from an existing custom protocol.

To open a sample protocol file1.

In the Home window, select File > Open > Protocol.
By default, Windows Explorer opens to the location of the CFX Maestro Dx SE Sample files folder.

2.

Open the Sample files folder. You see the following folders:nConventionalProtocols — contains example protocol files for traditional PCR analysis.

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Chapter 7 Creating Protocolsn

DataFiles — contains example data files that you can use to explore CFX Maestro Dx SE’s
features.

n

MeltCalibration — contains example protocol files for use with Bio-Rad’s Precision Melt
Analysis software.

nPlates — contains example plate files.

n

RealTimeProtocols – contains example protocol files for real-time PCR analysis.

3.

Open the protocol folder for the type of run you plan to perform, either ConventionalProtocols or
RealTimeProtocols.

4.

Select the protocol of choice and click Open.
The sample protocol opens in the Protocol Editor window.

5.

Select File > Save As and save the protocol with a new name or in a new folder.

To open an existing protocol
1.

In the Home window, do one of the following:nSelect File > Open > Protocol, navigate to and select the target protocol, and click Open.

n

Open the Startup Wizard and do one of the following:o

To edit the displayed protocol, click Edit Selected.

o

To edit another existing protocol, click Select Existing and navigate to the target file.

The protocol opens in the Protocol Editor window.
2.

Select File > Save As and save the protocol with a new name or in a new folder.

Setting Up a New Protocol
Tip: If your protocol file includes the required parameters (for example, if you are editing an existing
plate file) you can skip this section. Proceed to Adding Steps to a Protocol on page 102.
New protocol files require the following parameters:nBlock typen

Scan mode for the chosen block typen

Lid temperaturen

Sample volume

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Creating a Protocol in the Protocol Editor

Setting the Block Type
CFX Maestro Dx SE automatically calculates temperature increments for gradient steps based on theblock type.
Note: The plate type set in the Protocol Editor must be the same as the plate in the reaction module.

To set the block typeu

In the Protocol Editor window, select Tools > Gradient Calculator and choose the appropriate plate
type in the dropdown list that appears.

Selecting the Scan Mode for the Chosen Block TypeTo determine the run time for the protocol, select the target block type and scan mode.

To select the block type and scan modeu

In the Protocol Editor window, select Tools > Run time Calculator and choose the appropriate plate
type and scan mode in the dropdown list that appears.

Adjusting the Lid Temperature
CFX Maestro Dx SE sets the default lid temperatures as follows:n

96-well and deep well instruments — 105.0°C

n384-well instruments — 95.0°C

You can change the default settings or turn off the lid heater as necessary for the protocol.

To adjust the lid temperature
1.

In the Plate Editor window, select Settings > Lid Settings.
The Lid Settings dialog box appears.

2.

3.

Do one of the following:nSelect User Defined and enter a temperature value in the text box.

n

Select Turn Off Lid Heater.

Click OK to accept the changes and close the dialog box

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Chapter 7 Creating Protocols

Setting the Sample Volume
By default, CFX Maestro Dx SE sets the sample volume for each well to 25 µl. Sample volumes differ
depending on the type of block, for example:n0–50 µl for a 96-well blockn

0–30 µl for a 384-well block

The instrument uses one of two temperature control modes to determine when the sample reaches thetarget temperature in a protocol:nCalculated mode — when the sample volume is set to a non-zero volume appropriate for the block,the instrument calculates the sample temperature based on the sample volume. This is the standard
mode.

n

Block mode — when the sample volume is set to zero (0) µl, the instrument records the sample
temperature as the same as the measured block temperature.

To set the sample volume for a specific blocku

In the Plate Editor window, type the correct value in the Sample Volume text box on the toolbar.

Tip: You can change the default sample volume in the User Preferences dialog box. See Changing
the Default File Settings on page 79.

Adding Steps to a Protocol
To add a step to a protocol
1.

Open the protocol in the Protocol Editor window.

2.

Determine where to insert the new step. On the toolbar, select Before or After in the Step dropdown
list.

3.

On the graph, select the step before or after which you plan to insert the new step.

4.

In the left pane, click Insert Step.

5.

To change the temperature or hold time, click the default value on the graph or the protocol outline
and type a new value.

6.

(Optional) In the left pane, click Step Options to display the Step Options dialog box and modify theavailable options for the selected step.
Tip: You can access the Step Options dialog box on the right-click menu in either the graph paneor the protocol outline pane.

7.

Click OK and then click Yes to save changes to the protocol.

102 | CFX Maestro Dx SE Software

Creating a Protocol in the Protocol Editor

The Save As dialog box appears.
8.

In the Save As dialog box, type a name for the new protocol file and click Save.

Inserting a Gradient Step
To insert a gradient step
1.

Verify that the plate size for the gradient is the same as the block type of the instrument, 96-well, 384well, or deep well.

2.

If you have not yet done so, select the plate size for the gradient:
Select Tools > Gradient Calculator and choose the appropriate well type from the dropdown list.

3.

On the toolbar, select either Before or After from the Insert Step dropdown list.

4.

In the graph or outline pane, select the step before or after which you plan to insert the gradient step.

5.

In the left pane, click Insert Gradient. The new gradient step is highlighted in the graph and the outline
pane, for example:

The temperature of each row in the gradient appears in the Gradient table in the right pane.

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Chapter 7 Creating Protocols

6.

To edit the gradient temperature range, do one of the following:n

Click the default temperature in the graph or outline pane and enter a new temperature.

n

Click Step Options to enter the gradient range in the Step Options window.

nChange the Range value in the Gradient table.

7.

To edit the hold time, click the default time in the graphic or text view and enter a new time.

8.

Click OK and then Yes to save the changes.

Inserting a GOTO Step
Note: You cannot insert a GOTO step within a GOTO set; you cannot create nested GOTO loops.

To insert a GOTO step
1.

On the toolbar, select Before or After from the Insert Step dropdown list.

2.

In the graph, select the step before or after which you plan to insert the GOTO step.

3.

In the left pane, click Insert GOTO.

4.

To edit the GOTO step number or number of GOTO repeats, select the default number in the graphor outline pane and enter a new value.

5.

Click OK and then Yes to save the changes.

Inserting a Melt Curve Step
Tip: You cannot insert a melt curve step inside a GOTO loop.
Note: The melt curve step includes a 30 sec hold at the beginning of the step that is not shown in the
protocol.

To insert a melt curve step
1.

On the toolbar, select Before or After from the Insert Step dropdown list.

2.

In the graph, select the step before or after which you plan to insert the melt curve step.

104 | CFX Maestro Dx SE Software

Creating a Protocol in the Protocol Editor

3.

In the left pane, click Insert Melt Curve. The new melt curve step is highlighted in the graph and theoutline pane, for example:

4.

To edit the melt temperature range or increment time, select the default number in the graph oroutline pane and enter a new value.

5.

Click OK and then Yes to save the changes.

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Chapter 7 Creating Protocols

Adding or Removing a Plate Read Step
Tip: After you add a plate read command to a step, the button changes to Remove Plate Read when
you select the step.

To add a plate read to a step
1.

On the toolbar, select Before or After from the Insert Step dropdown list.

2.

On the graph, select the step before or after which you plan to insert the plate read step.

3.

In the left pane, click Add Plate Read to Step to add a plate read to the selected step.

4.

Click OK and then Yes to save the changes.

To remove a plate read from a stepu

On the graph, select the step that contains the plate read and click Remove Plate Read in the leftpane.

Changing Step Options
To change step options for a selected step
1.

Select the target step in the graph or outline pane.

2.

In the left pane, click Step Options to open the Step Options dialog box.
Alternatively, right-click the target step in either pane and select Step Options in the menu thatappears.

3.

To add, modify, or remove options:nEnter a value in the appropriate text box.

n

Edit a value in the specific text box.

n

Select or clear a checkbox.

4.

Click OK to save the changes and close the Step Options dialog box.

5.

Click OK and then Yes to save the protocol.

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Creating a Protocol in the Protocol Editor

Deleting a Step
Important: You cannot undo this function. Take care when deleting steps.

To delete a step in the protocol
1.

Select the step in the graph or outline pane.

2.

In the left pane, click Delete Step to delete the selected step.

3.

Click OK and then Yes to save the protocol.

Copying, Exporting, or Printing a Protocol
To copy a protocoluRight-click the protocol outline and select Copy Protocol.
You can paste the outline into a .txt, .xls, .doc, or .ppt file.

To export a protocol
1.

Right-click the protocol outline and select Export Protocol.
The Save As dialog box appears.

2.

(Optional) In Windows Explorer, navigate to a folder in which to save the protocol file.

3.

In File name, type a name for the exported protocol file.

4.

Click Save.

To print a protocoluRight-click the protocol outline and select Print.
You can print the protocol outline to your default printer.

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Chapter 7 Creating Protocols

Creating a Protocol with the Protocol AutoWriter
Important: Bio-Rad does not guarantee that running a protocol created with the Protocol AutoWriter
will always result in a PCR product.
CFX Maestro Dx SE’s Protocol AutoWriter automatically generates cycling protocols based on thefollowing input parameters:nAmplicon length — the expected length of the PCR productnAnnealing temperature — the reaction Ta for the primers being used
If the Ta is unknown, you can use the Ta calculator to automatically calculate it based on your primer
sequences.
Note: The Ta is adjusted from the primer melting temperature (Tm) information that is based on
the selected enzyme and the protocol speed.

nEnzyme type — the DNA polymerase enzyme (iTaq, iProof DNA polymerase, or Other)If you use an enzyme other than iTaq or iProof DNA polymerase, you can enter additional
information, including the gradient range, hot-start activation time (in sec), and the final extension
time (in sec).

n

Run speed — the reaction speed (standard, fast, or ultrafast)
The Protocol AutoWriter optimizes the protocol depending on the selected speed setting. The total
run time is determined by the number of steps and cycles, the incubation time at each step, and thetime it takes to reach uniformity at the target temperature.

Using parameters that you enter and standard PCR guidelines, the Protocol AutoWriter automaticallygenerates a customized PCR protocol with hot start, initial denaturation, annealing, and extension steps.
You can then view a graphical representation of the suggested protocol and edit, run, or save the protocol.

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Creating a Protocol with the Protocol AutoWriter

To create a new protocol using CFX Maestro Dx SE’s Protocol AutoWriter
1.

In the Home window, select Tools > Protocol AutoWriter.
The Protocol AutoWriter dialog box appears.

2.

In the Enter Target Values/Enzyme section, do the following:nEnter the annealing temperature (Ta) for the primers, if known.
Tip: See Using the Ta Calculator on page 110 for more information.
Note: For information about the calculations used in the Ta Calculator, see Breslauer et al.
1986.

n

Enter the amplicon length in base pairs (bp).

nSelect an enzyme type from the list of options (iTaq DNA polymerase, iProof DNA polymerase,or Other).
Tip: If you select Other as the enzyme type, the parameters in the Additional Parameters(Optional) section become active.

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Chapter 7 Creating Protocols

3.

If you selected Other as the enzyme type, you can add any or all of the following parameters to theprotocol:n

Gradient rangen

Hot start activation temperaturenFinal extension time

4.

In the Type section, move the sliding bar to select a protocol speed (Standard, Fast, or Ultrafast).
CFX Maestro Dx SE adjusts the total run time.

5.

Select the type of PCR to perform (Real-time PCR is the default).
With real-time PCR, CFX Maestro Dx SE adds a plate read step to collect fluorescence data.

6.

In the Preview section, review the protocol. You can make changes as needed.

7.

Do one of the following:n

Click OK to save the new protocol. After saving it, the protocol opens in the Startup Wizard. Click
Edit Selected to make any changes to the protocol. For example, you might need to change thelid temperature and sample volume.

n

Click Cancel to close the window without saving the protocol.

Using the Ta Calculator
When the annealing temperature for the primer is unknown, you can use the Ta Calculator to calculate thevalue. You can use the value in the Protocol AutoWriter or in the Protocol Editor to create your protocol.

About the Ta Calculator
The Ta Calculator calculates the Tm value for each primer as well as the Ta value for the protocol atstandard speed.
The Ta for the protocol is based on the average primer Tm values with the following rules applied:n

If the difference between the primer Tm values is >4°C, the Ta = (lower of the two primer Tm values +2) – 4°Cn

If the difference between the Tm values is ≤4°C, the Ta = (average of the primer Tm values) – 4°C

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Using the Ta Calculator

Base Pair Counting Method
For each primer, the Ta Calculator uses the base pair counting method for sequences of 14 base pairs(bp) or fewer.
Tm = ((w*A + x*T) * 2) + ((y*G + z*C) * 4)
where w, x, y, and z are the numbers of the bases A, T, G, and C in the sequence, respectively.

Nearest Neighbor Method
For sequences longer than 14 bp, the nearest neighbor method is used. In the nearest neighbor method,
the melting temperature calculations are based on the thermodynamic relationship between entropy(order or a measure of the randomness of the oligonucleotide), enthalpy (heat released or absorbed by
the oligonucleotide), free energy, and temperature.
∆H = ∆G + T* ∆S
where:n∆H = Enthalpy value, Cal/Mole*Kn

T = temperature, Kelvinn∆S = Entropy value, Cal/Mole*Kn∆G = Gibbs free energy in Cal/Mole*K

The change in entropy and enthalpy is directly calculated by summing the values for nucleotide pairs
shown in Table 8 (Breslauer et al. 1986).
The relationship between the free energy and the concentration of reactants and products at equilibrium isgiven by:
∆G = R*T*ln ((DNA * Primer)/(DNA + Primer))where R is the gas constant (1.986 Cal/Mole*K).
Substituting G in the two equations and solving for T gives
T = ∆H/(∆S + R*Ln((DNA * Primer)/(DNA + Primer)))
assuming that the concentration of DNA and DNA-primer complex are equal.
It has been determined empirically that there is a 5 kcal free energy (3.4 kcal) (Sugimoto et al. 1996)
change during the transition from single-stranded to B-form DNA. This is presumably helix initiationenergy. Finally, adding an adjustment for salt gives the equation that the Ta calculator uses:
T = (∆H – 5(KCal/K*Mole))/(∆S + (R * ln(1/(primer)))) + 16.6 log10 (SaltMolarity)

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Chapter 7 Creating Protocols

No adjustment constant for salt concentration is needed, since the various parameters were determinedat 1 M NaCl, and the log10 of 1 is zero.
The thermodynamic calculations assume that annealing occurs at pH 7.0. The Tm calculations assume
that the sequences are not symmetrical and contain at least one G or C.
The oligonucleotide sequence should be at least 14 bases long to give reasonable Tm values. Less than
14 bases use the base pair counting method (see Table 8 that follows).
Table 8. Breslauer interaction constants

Interaction

∆H

∆S

∆G

AA

TT

9.1

24

1.5

AT

TA

8.6

23.9

1.5

AC

TG

6.5

17.3

1.3

AG

TC

7.8

20.8

1.6

TA

AT

6

16.9

0.9

TT

AA

9.1

24

1.9

TC

AG

5.6

13.5

1.6

TG

AC

5.8

12.9

1.9

CA

GT

5.8

12.9

1.9

CT

GA

7.8

20.8

1.6

CC

GG

11

26.6

3.1

CG

GC

11.9

27.8

3.6

GA

CT

5.6

13.5

1.6

GT

CA

6.5

17.3

1.3

GC

CG

11.1

26.7

3.1

GG

CC

11

26.6

3.1

112 | CFX Maestro Dx SE Software

Using the Ta Calculator

Using the Ta Calculator
To use the Ta Calculator
1.

To open the Ta Calculator, do one of the following:n

If you are currently in the Protocol AutoWriter, click Ta Calculator.

n

In the Home window, select Tools > Ta Calculator.

The Ta Calculator dialog box appears.

2.

In the Forward Primer text box, type or paste the forward primer sequence.
Tip: You can also use the A, T, G, C buttons on the left side of the dialog box to enter thesequence.

3.

Type or paste the reverse primer sequence in the Reverse Primer text box.

4.

Click Calculate.

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Chapter 7 Creating Protocols

The Ta Calculator calculates and displays the Tm of each primer and the average Tm and Ta values,for example:

If the primer Tm values are more than 4°C apart, the Protocol AutoWriter uses the lower primer Tmvalue + 2°C as a basis for calculating the Ta value, which you can further modify by changing the
enzyme and reaction speed.
The Ta Calculator generates an annealing temperature for standard speed with iTaq DNA
polymerase. When using a different enzyme, the speed settings automatically adjust the Ta.
5.

Do one of the following:n

If you opened the Ta Calculator from the Protocol AutoWriter, click OK. You return to the Protocol
AutoWriter. The annealing temperature is automatically modified.

n

If you opened the Ta Calculator from the Tools menu, record the calculations and click Cancel toclose the calculator.

114 | CFX Maestro Dx SE SoftwareChapter 8 Preparing Plates
A plate file contains information about run parameters such as scan mode, fluorophores, and wellcontents. After the run, CFX Maestro Dx Software, Security Edition links the well contents to thefluorescence data collected during the run and applies the appropriate analysis in the Data Analysis
window. For example, wells loaded with standard sample type are used to generate a standard curve.
CFX Maestro Dx SE provides two options for creating plates: The Plate Editor for real-time PCR runs andthe Setup Wizard for normalized gene expression analysis.
The Plate Editor includes the following features:nStandard fluorophores and sample types to assign to plate wellsnAbility to set reference target and control sample for gene expression analysisnAbility to edit plate setup before, during, or after a runnAbility to save plate files for reusenAbility to print the plate file to a default printerThe Setup Wizard guides you through creating a plate layout for normalized gene expression analysis.
You can use the Setup Wizard before, during, or after a run.

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Chapter 8 Preparing Plates

Plate Editor Window
You use the Plate Editor to create custom plates or modify existing plates.

LEGEND1.

The menu bar provides quick access to File and Settings menu commands as well as plate
editing tools options.

2.

The toolbar provides quick access to important plate loading functions.

3.

The main pane displays the plate outline and the plate options as you apply them.

4.

The right pane displays options that you use to customize your plate.

5.

The bottom pane displays the plate type and provides quick access to viewing options.

File Menu Commands
Save — saves the plate data file in the location specified in the File tab in the User Preferences dialog
box. See Changing the Default File Settings on page 79 for more information. This menu item is available
only when creating a new plate file.
Save As — saves the open plate data file with a new name that you supply. This menu item is available
only when creating a new plate file.
File Passwords — enables users to set their file save and file open passwords.

116 | CFX Maestro Dx SE SoftwarePlate Editor Window

Extract Plate — opens a dialog box in which you can extract/save the plate file (.pltd). This menu item isavailable only when viewing or editing an existing plate file.
Print — prints the open plate data file.
Close — closes the Plate Editor.

Edit Menu Commands
Undo — reverts a change to a plate file until the plate file is saved.
Redo — reverses the most recent Undo action unless the plate file has been saved.

Settings Menu Commands
Plate Size — opens a dialog box from which you can select a plate size for the run.
Note: The plate size must be the same as the block size on the instrument on which the run isperformed.
Choose 96-well for:n

CFX Opus 96 Dxn

CFX Opus Deepwell Dx

Choose 384-well for:n

CFX Opus 384Dx

Plate Type — allows you to choose the type of wells in the plate that holds your samples, either BR Whiteor BR Clear. For accurate data analysis, the plate type selected must be the same as the plate type usedin the run.
Note: You must calibrate new plate types. See Calibrating New Dyes on page 72 for more
information.
Number Convention — allows you to select or deselect the option to display units in scientific notation.
The default is to display units in scientific notation.
Units — allows you to choose the units to show in the spreadsheets when performing quantification of
unknowns vs. a standard curve.

Editing Tools Menu Commands
Setup Wizard — opens the Setup Wizard, in which you can define layout and analysis parameters for thecurrent plate. You can use the Setup Wizard before, during, or after a run has completed.

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Chapter 8 Preparing Plates

Spreadsheet View/Importer — opens the View dialog box, which displays the plate layout as a templatein spreadsheet format. You can use this dialog box to export or import plate template data in .csv format.
Flip Plate — flips the plate contents 180o.

Toolbar Commands
Reverts a change to a plate. CFX Maestro Dx SE supports up to ten undo
actions
Reverses the most recent Undo action. CFX Maestro Dx SE supports up
to ten redo actions.
Saves the current plate file.

Displays a dropdown list from which you can increase or decrease themagnification of the plate view.
Displays a dropdown list from which you can select a scan mode, which
instructs the instrument from which channels to collect fluorescence data
during a run.
Opens the Well Groups Manager, which you can use to create wellgroups for the current plate.
Displays a dialog box in which you can choose the colors and symbols forthe amplification traces.
Opens the View dialog box, which displays the plate layout as a templatein spreadsheet format. You can use this dialog box to export or importplate template data in .csv format.
Opens the Setup Wizard, in which you can define the layout and analysis
parameters for the current plate. You can use the Setup Wizard before,
during, or after a run.
Opens the Plate tab in the User Preferences dialog box, in which you candefine plate layout parameters and create or delete target, sample, andbiological group names. Changes that you make in the Plate tab are
available the next time you open the Plate Editor.
Displays the necessary steps for setting up a plate and loading the wells.

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Creating a Plate File Using the Plate Editor

Creating a Plate File Using the Plate Editor
Using the Plate Editor, you can create custom plate files. You can also edit and save previously saved
plate files or sample plate files shipped with the CFX Opus Dx system.
To create a new plate file, perform the following:nOpen a plate file in the Plate Editor.

nSelect the plate type.
Note: The plate type for the plate file must be the same as the plate in the reaction module.

nSelect the scan mode to use in the protocol.

nSelect the fluorophores to use in the plate.

n

Select the sample type, targets, and samples.

nSelect technical replicates, if appropriate.

n

Save the plate layout.

Tip: To create a new plate from previously saved or sample plate files, see Opening an Existing Plate
File in the Plate Editor on page 121.

Opening a New Plate File in the Plate Editor
CFX Maestro Dx SE offers multiple options to open a new plate file:n

From the Home windown

From the Startup Wizard dialog box

nFrom the Run Setup dialog box

To open a new plate file from the Home windowuSelect File > New > Plate.
The Plate Editor window opens displaying the default plate file for the selected instrument.
Tip: For information about setting your default plate file, see Changing the Default File Settings
on page 79.

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Chapter 8 Preparing Plates

To open a new plate file from the Startup Wizard
1.

In the Home window, do one of the following to open the Startup Wizard if it is not in view:nSelect View > Startup Wizard.

n

Click Startup Wizard on the toolbar.

2.

If necessary, select the instrument type from the dropdown list.

3.

To create a new plate, click User-defined as the run type.
The Run Setup dialog box opens displaying the Protocol tab.

4.

Click the Plate tab and click Create New.
The Plate Editor window opens displaying the default plate layout for the selected instrument.

To open a new plate file from the Run Setup dialog box
1.

In the Home window, do one of the following to open the Run Setup dialog box:n

Select Run > User-defined Run.

n

Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab.
2.

To create a new plate, click the Plate tab and click Create New.
The Plate Editor window opens displaying the default plate layout for the selected instrument.

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Creating a Plate File Using the Plate Editor

Opening an Existing Plate File in the Plate Editor
CFX Maestro Dx SE provides sample plate files that you can edit and save as a new plate. You can also
create a new plate file from a previously saved plate file.

To open a sample plate file1.

In the Home window, select File > Open > Plate.
Windows Explorer opens to the location of the CFX Opus Dx system Sample files folder.

2.

Open the Sample files folder, and then open the Plates folder.

3.

Select a plate file and click Open.
The sample plate file opens in the Plate Editor window.

4.

Select File > Save As and save the plate file with a new name or in a new folder.

To open a previously saved plate file1.

In the Home window, do one of the following:nSelect File > Open > Plate, navigate to and select the target plate, and click Open.

n

Open the Startup Wizard and do one of the following:o

To edit an existing plate file, click Select Existing and navigate to the target file.

oTo edit the displayed plate file, click Edit Selected.

The target plate opens in the Plate Editor window.
2.

Select File > Save As and save the plate file with a new name or in a new folder.

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Chapter 8 Preparing Plates

Setting Up a New Plate File
Tip: If your plate file includes the required parameters (for example, if you are editing a sample orexisting plate file) you can skip this section. Proceed to Assigning Optional Parameters to the Plate
File on page 129.
New plate files require the following parameters:nPlate sizenPlate typen

Scan moden

One fluorophore (dye)nOne sample type

Selecting the Plate Size and Type
Important: You must select a plate size during plate setup. You cannot change the plate size during
or after a run.
The software applies the plate size and type to all the wells during the run. Ensure that the plate size
selected is the same as the plate you will use in the run.
Bio-Rad’s CFX Opus Dx systems are factory-calibrated for many fluorescent dye and plate combinations.
Calibration is specific to the instrument, dye, and plate type. Ensure that the fluorophore you plan to use iscalibrated for the plate type you select.
Tip: To calibrate a new combination of dye and plate type on an instrument, select Tools > Dye
Calibration Wizard. For information about calibrating dyes and plate types, see Calibrating New Dyes
on page 72.

Selecting Scan Mode
The CFX Opus 96 Dx and CFX Opus Deepwell Dx systems excite and detect fluorophores in fivechannels (plus FRET). The CFX Opus 384 Dx system excites and detects fluorophores in four channels(plus FRET). All systems use multiple data acquisition scan modes to collect fluorescence data during arun.
CFX Maestro Dx SE provides three scan modes:n

All ChannelsoScans channels 1 through 5 on CFX Opus 96 Dx and CFX Opus Deepwell Dx systemsoScans channels 1 through 4 on CFX Opus 384 Dx systems

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Creating a Plate File Using the Plate Editornn

SYBR®/FAMoScans only channel 1oProvides a fast scan

FREToScans only the FRET channeloProvides a fast scan

Selecting Fluorophores
Important: Before beginning the run, CFX systems verify that the fluorophores you specified in theplate are calibrated on that instrument. You cannot run a plate if it includes fluorophores that have notbeen calibrated on that instrument.
You must load at least one fluorophore to the plate layout before the run. You can add as many
fluorophores as necessary at this time, but the plate must contain at least one fluorophore. The selected
fluorophores appear as options for targets in Target Names.
You use the Select Fluorophores dialog box to load fluorophores (or plate dyes) into the Plate Editor wellloading controls. The fluorophores that appear in the Select Fluorophores dialog box depend on the scanmode you select:n

All Channels
All available fluorophores appear.
Tip: You can add as many fluorophores as necessary, but you can load only one fluorophore perchannel in each well.

n

SYBR®/FAM
Only channel 1 fluorophores appear.

n

FRET
Only the channel 6 fluorophore appears.
Tip: The channel 6 FRET fluorophore appears only when FRET is the selected scan mode. It is
not available for All Channels scan mode.

Note: You cannot directly add fluorophores to or remove them from the Select Fluorophore dialog
box. You must calibrate new fluorophores on an instrument using the Dye Calibration Wizard. Aftercalibration, the new fluorophore is automatically added to this list. For more information, see
Calibrating New Dyes on page 72.

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Chapter 8 Preparing Plates

Selecting Sample Types
Important: You must select at least one sample type to assign to the plate wells before the run.
CFX Maestro Dx SE offers five sample types:nUnknownnStandardn

NTC (no template control)nPositive Control

nNegative Controln

NRT (no reverse transcriptase)

You assign the sample types to the plate wells.

Setting Up a New Plate
To set up a new plate
1.

Open a new plate in the Plate Editor window.

2.

To set the plate size, select Settings > Plate Size and select the appropriate plate size from thedropdown menu.

3.

To set the plate type, select Settings > Plate Type and select either BR White or BR Clear from thedropdown menu.

4.

Optionally, from the Settings menu you can change the number convention and the display units:n

To change the number convention, select Settings > Number Convention and select Scientific
Notation.
Tip: Scientific Notation is selected by default. In this case, selecting Scientific Notation
clears the default and sets the number convention to standard form.

n
5.

To change the display units, select Settings > Units and select a new unit value.

To set the scan mode, select the appropriate scan mode from the Scan Mode dropdown list in the
Plate Editor window toolbar.

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Creating a Plate File Using the Plate Editor

6.

Select the requisite fluorophores for the plate:a.

In the right pane, click Select Fluorophores.
The Select Fluorophores dialog box appears. You see the fluorophores available for the type ofscan mode you selected in Step 5, for example:b.

To select a fluorophore, click its Selected checkbox.
Tip: To remove a fluorophore from the list, clear its Selected checkbox.

c.

To change the display color of the fluorophore, click its Color box.
Note: The color you select represents the fluorophore in both the Plate Editor window andthe Data Analysis charts.

d.

In the Color dialog box, select the color that you want or click Define Custom Colors and create a
new color to represent the fluorophore.

e.

Click OK to save the changes and exit the Select Fluorophores dialog box.

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Chapter 8 Preparing Plates

7.

You must select at least one well in which to load a sample type. By default, well A1 is selected.
In the plate pane, do one of the following:n

To load multiple adjacent wells, click a well and drag to the target well.

n

To load multiple nonadjacent wells, hold the Control key and click each well.

n

To load an entire column with the same sample type, click the column number.

n

To load an entire row, click its row number.

n

To load the whole plate, click the upper left corner of the plate.

For example:

8.

Assign a sample type to the selected well or wells from the Sample Type dropdown menu.

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Creating a Plate File Using the Plate Editor

9.

Assign at least one fluorophore to all wells that contain a sample type. You can assign more than one
fluorophore to a well or group of wells.
Note: You can assign only one fluorophore per channel. You cannot assign more than one
fluorophore from the same channel to the same well.
Tip: You can associate a target with the fluorophore, or you can assign only the fluorophore to
the well at this time and associate a target to the fluorophore after you run the experiment.
n

To assign only a fluorophore to the selected wells, in the Target Names section in the right paneselect the Load checkbox for the specific fluorophore.

n

To associate a target with a fluorophore, in the Target Names section select a target name fromthe dropdown list for the specific fluorophore. The software automatically selects its Loadcheckbox.

10. For wells containing a Standard sample type, you must load a concentration. Each well can have a
different concentration value. By default, CFX Maestro Dx SE loads a concentration of 1.00E+06 to
all wells with a Standard sample type. You can change the value if necessary.
a.

In the plate pane, select a Standard well or group of wells.

b.

In the Concentration section, click Load to load the value to the selected well or wells.

c.

(Optional) To load another concentration, type the new value in the Concentration text box andpress enter.

d.

Perform this step for all wells with sample type Standard.
Tip: To load the same concentration to all Standard wells, ensure that <All> appears in the
dropdown list below the Concentration value. To load the same concentration value to allwells with a specific fluorophore, click the dropdown list and select the fluorophore.

11. Click OK to save the new plate.

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Chapter 8 Preparing Plates

Right-Click Menu Items for the Plate Editor Tool
Table 9 lists the menu items available in the Plate Editor tool when you any right-click on any well in thetool. This menu also appears in the Spreadsheet View/Importer.
Table 9. Right-click menu items in the Plate Spreadsheet View/Importer tool

Item

Function

Copy

Copies the entire spreadsheet.

Copy as Image

Copies the spreadsheet as an image file.

Print

Prints the spreadsheet.

Print Selection

Prints only the selected cells.

Export to Excel

Exports the file to an Excel spreadsheet.

Export to CSVExports the file as a .csv file.

Export to Xml

Exports the file as an .xml file.

Export to Html

Exports the file as an .html file.

Find

Searches for specific text.

Sort

Sorts the spreadsheet by selecting up to three columns of data in
the Sort window.

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Assigning Optional Parameters to the Plate File

Assigning Optional Parameters to the Plate File
A plate file contains information about the contents of each well loaded with sample for a run. After therun, CFX Maestro Dx SE links the well contents to the fluorescence data collected during the protocol andapplies the appropriate analysis in the Data Analysis window.
In CFX Maestro Dx SE, you can assign parameters to each well in your plate before, during, or even after
you run experiments. You can assign the parameters to an existing plate file or to a new plate file. These
parameters include:nTarget names — the target or targets of interest (genes or sequences) in each loaded well.

nSample names — the identifier or condition that corresponds to the sample in each loaded well,such as mouse1, mouse2, or mouse3.

nBiological groups — the identifier or condition that corresponds to a group of wells, such as 0Hr,
1Hr, or 2Hr.
Tip: Target names, sample names, and biological groups must be the same between wells tocompare data in the Gene Expression tab of the Data Analysis window. Each name must containthe same capitalization, punctuation, and spacing. For example, “Actin” is not the same as“actin,” “2Hr” is not the same as “2 hr,” and “Mouse 1” is not the same as “mouse1.” To ensure
naming consistency, enter the names in the Libraries section in User > User Preferences >Plate, available on the Home window.

nTechnical replicates — each well that is used to analyze the same combination of sample andtarget; that is, replicate qPCR reactions.

nDilution series — the amount to change the concentration of the Standard sample type within areplicate group to create standard curve data to analyze.

Assigning a Target to Wells
Tip: You can assign the same target name to a single or multiple wells. You can also assign multiple
targets to the same well.
Important: Clicking OK after you assign a target saves the changes and disables Undo on the Plate
Editor toolbar. Take care when clicking OK.

To assign a target to a well or group of wells
1.

In the Plate Editor, ensure that the well or group of wells have been assigned a sample type.
See Selecting Sample Types on page 124 for information about assigning sample types to wells.

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Chapter 8 Preparing Plates

2.

In the plate pane, select the well or group of wells:nTo select a single well, click the well.

n

To select multiple adjacent wells, click a well and drag to the target well.

n

To select multiple nonadjacent wells, hold the Control key and click each well.

n

To select an entire column with the same sample type, click the column number.

n

To select an entire row, click its row number.

3.

In the right pane, select a name from the Target Name dropdown list for each selected fluorophore.

4.

Repeat Step 3 for each well or group of wells to which you must assign a target.
Tip: You can assign the same or a different target name for each selected fluorophore.

5.

Click OK to accept the changes and save the plate.
Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you click OK
to accept the changes.

To remove a target nameu

To remove a target name from the selected well or group of wells, clear its Load checkbox.
Important: Removing a target name from a well also removes its associated fluorophore. Takecare when removing a target name from a well.

To add a target name to the listu

To add a target name to the dropdown list, do one of the following:nType a name in the Target Name dropdown list and press Enter.
Tip: Target names that you add to one list appear in all other target lists.

n

Click the green + symbol to the right of the dropdown list, type a name for the target and press
Enter.

n

Click User Preferences on the toolbar and add the name to the Target Names library in the Platetab.

130 | CFX Maestro Dx SE Software

Assigning Optional Parameters to the Plate File

Important: Target names that you add in the dropdown list are available only for the current
plate, and only if you assign the name to a well and save the plate layout. If you do not assign thename to a well and save the plate layout, the name is not saved and is not available for futureuse. To permanently add a target name, also add it to the Target Names library using the User
Preferences dialog box. Names that you add to the library are available after you open the Plate
Editor again. See Setting Default Plate Parameters on page 81 for more information.

To delete a target name from the list
1.

Click User Preferences on the toolbar.
The User Preferences dialog box appears, displaying the Plate tab.

2.

In the Target Names library in the Plate tab, select the name to delete and press the Delete key.

3.

Click OK to save changes and exit the User Preferences dialog box.
Important: You cannot delete target names that you saved with a plate file. Custom names thatyou add to the Target Names dropdown list and do not use and save with the plate areautomatically removed from the list. Names that you delete from the Target Names Library arepermanently removed from the software and are no longer available to users. Take care whendeleting target names.

Assigning a Sample Name to Wells
Note: To assign a sample name, you must assign the selected wells at least one fluorophore. If theselected wells are not assigned a fluorophore, the Sample Names dropdown list is disabled. See
Assigning a Target to Wells on page 129 for information about assigning fluorophores.
Tip: You can assign only one sample name to each well or group of wells.

To assign a sample name to a well or group of wells
1.

In the Plate Editor, ensure that the well or group of wells has been assigned a fluorophore.

2.

In the plate pane, select the well or group of wells.

3.

In the right pane, select a name in the Sample Names dropdown list.
The software automatically selects its Load checkbox.

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Chapter 8 Preparing Plates

4.

Repeat Step 3 for each well or group of wells to which you must assign a sample name.

5.

Click OK to accept the changes and save the plate.
Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you click OK
to accept the changes.

To remove a sample nameu

To remove a sample name from a selected well or group of wells, clear its Load checkbox.

To add a sample name to the listu

To add a sample name to the dropdown list, do one of the following:nType a name in the Sample Names dropdown list and press Enter.

n

Click the green + symbol to the right of the dropdown list and type a name for the sample.

nClick User Preferences on the toolbar and add the name to the Sample Names library in the
Plate tab.

Important: Sample names that you add in the dropdown list are available only for the current
plate, and only if you assign the name to a well and save the plate layout. If you do not assign thename to a well and save the plate layout, the name is not saved and is not available for futureuse. To permanently add a sample name, also add it to the Sample Names library using the User
Preferences dialog box. Names that you add to the library are available after you open the Plate
Editor again. See Setting Default Plate Parameters on page 81 for more information.

To delete a sample name from the list
1.

Click User Preferences on the toolbar.
The User Preferences dialog box appears, displaying the Plate tab.

2.

In the Sample Names library in the Plate tab, select the name to delete and press the Delete key.

3.

Click OK to save changes and exit the User Preferences dialog box.
Important: You cannot delete sample names that you have saved with a plate file. Custom
names that you add to the Sample Names list and do not use and save with the plate areautomatically removed from the dropdown list. Names that you delete from the Sample Names
Library are removed from the software and are no longer available to users. Take care whendeleting sample names.

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Assigning Optional Parameters to the Plate File

Assigning Biological Groups to Wells
Note: To assign a biological group, you must assign the selected wells at least one fluorophore.
Assigning a fluorophore enables the Biological Groups dropdown list. See Assigning a Target toWells on page 129 for information about assigning fluorophores.
Tip: You can assign one biological group to each well or group of wells.

To assign a biological group to a well or group of wells
1.

In the Plate Editor, ensure that the well or group of wells has been assigned a fluorophore.

2.

In the plate pane, select the well or group of wells.

3.

In the right pane, make a selection from the Biological Group dropdown list.
CFX Maestro Dx SE automatically selects its Load checkbox.

4.

Repeat Step 3 for each well or group of wells to which you must assign a biological group.

5.

Click OK to accept the changes and save the plate.
Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you click OK
to accept the changes.

To remove a biological groupu

To remove a biological group from the selected well or group of wells, clear its Load checkbox.

To add a biological group to the listu

To add a biological group to the dropdown list, do one of the following:nType a name in the Biological Group dropdown box and press Enter.

n

Click the green + symbol to the right of the dropdown list and type a name for the biologicalgroup.

n

Click User Preferences on the toolbar and add the name to the Biological Group Names library in
the Plate tab.

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Chapter 8 Preparing Plates

Important: Biological group names that you add in the dropdown list are available only for thecurrent plate, and only if you assign the name to a well and save the plate layout. If you do not
assign the name to a well and save the plate layout, the name is not saved and is not available
for future use. To permanently add a biological group name, also add it to the Biological Group
Names library using the User Preferences dialog box. Names that you add to the library areavailable after you open the Plate Editor again. See Setting Default Plate Parameters on page
81 for more information.

To delete a biological group name from the list
1.

Click User Preferences on the toolbar.
The User Preferences dialog box appears, displaying the Plate tab.

2.

In the Biological Group Names library in the Plate tab, select the name to delete and press the Delete
key.

3.

Click OK to save changes and exit the User Preferences dialog box.
Important: You cannot delete biological group names that you saved with a plate file. Custom
names that you add to the Biological Group Names dropdown list and do not use and save withthe plate are automatically removed from the list. Names that you delete from the BiologicalGroup Names Library are permanently removed from the software and are no longer available tousers. Take care when deleting biological names.

To view all biological groups on the plateu

Click Show Biological Groups to view all biological groups on the plate.

Each group is identified by a specific color and the Show Biological Groups button changes to Hide
Biological Groups.
Click Hide Biological Groups to clear the color in the wells. Alternatively, you can click any well in theplate to hide biological groups.

Assigning Technical Replicate Numbers to Wells
Important: To assign technical replicate numbers, the selected wells must contain identical wellcontents. That is, the selected wells must have the same sample type and fluorophore. If appropriate,134 | CFX Maestro Dx SE Software

Assigning Optional Parameters to the Plate Filethey must also be assigned the same target and sample names and the same biological group. If they
are not the same, CFX Maestro Dx SE does not enable this option.

To assign technical replicate numbers to a group of wells
1.

In the Plate Editor, ensure that the contents of the group of wells are identical.

2.

In the plate pane, select the target group of wells.

3.

To assign the same replicate number to all selected wells, in the Replicate # section in the right panetype the replicate number in the box and select Load.

4.

(Optional) To apply a replicate series to a set of selected wells:a.

Click Technical Replicates. The Replicate # section changes to display the following options:nReplicate size — a number that represents the number of wells in each group of replicatesnStarting replicate # — the first number in the replicate series for the selected group of
replicates

Note: By default, CFX Maestro Dx SE displays the starting replicate number as one number
greater than the last technical replicate number assigned in the plate. For example, if thelast technical replicate number in the plate is five, the next starting number is six. You canchange the starting number to any number that is not already assigned.
nb.
5.

Loading direction (Horizontal or Vertical)

Click Apply to apply the parameters to the series and return to the Replicate # display.

Click OK to accept the changes and save the plate.

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Chapter 8 Preparing Plates

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you click OK
to accept the changes.

To remove a well from a replicate seriesu

Select the well or group of wells to be removed and clear the Replicate # Load checkbox.
Alternatively, you can click Clear Replicate # to clear the replicate number from a selected well orgroup of wells.

To view all technical replicates on the plateu

Click Show Technical Replicates to view all technical replicates on the plate.
Each group is identified by a specific color and the Show Technical Replicates button changes to
Hide Technical Replicates.
Click Hide Technical Replicates to clear the color in the wells. Alternatively, you can click any well in
the plate to hide technical replicates.

Assigning a Dilution Series to Standard Sample Types
As previously mentioned, all wells with the sample type Standard must be assigned a concentration value.
You can assign a dilution series to multiple wells with the sample type Standard.
Note: In order to assign a dilution series to a group of wells, the wells must be included in a technicalreplicate series. See Assigning Technical Replicate Numbers to Wells on page 134 for information
about adding wells to a replicate series.

To assign a dilution series to a group of Standard sample wells
1.

In the Plate Editor, ensure the following requirements are met:n

The sample type for the group of wells is Standard.

nAll wells in the group are assigned at least one fluorophore and they all contain the same
fluorophores.

n

All wells in the group are included in the same technical replicate series.
Note: CFX Maestro Dx SE enables the Dilution Series option only when all selected wells
meet these criteria.

2.

In the plate pane, select the target group of wells.

3.

In the Concentration section in the right pane, click Dilution Series. The Concentration sectionchanges to display the following options:

136 | CFX Maestro Dx SE Software

Assigning Optional Parameters to the Plate FilenStarting concentration — the concentration value from which the series startsnReplicates from and to — the replicates in the series to which the dilution factor will be appliednDilution factor — the amount to change the concentration within each replicate group4.

Set the values for the options or accept the defaults.

5.

By default, the dilution series decreases by the dilution factor. Select Increasing to increase thedilution series.

6.

(Optional) By default, the dilution factor applies to all fluorophores in the replicate series. If yourseries contains more than one fluorophore and you want to apply the dilution to a single fluorophore,
select it from the dropdown list.

7.

Click Apply to apply the dilution series to the group of wells and return to the Concentration view.

8.

Click OK to accept the changes and save the plate.

Copying Well Contents into Another Well
You can copy the contents of a well and paste it into a single well or multiple wells. However, you can copy
the contents of only a single well. You cannot select multiple wells and copy their contents.

To copy well contents into another well1.

In the plate pane, select the well to be copied.

2.

Right-click the well and select Copy Well.

3.

Select the well or wells into which content is to be pasted:4.

n

To select a single well, click on the well.

n

To select multiple adjacent wells, click a well and drag to the target well.

n

To select multiple nonadjacent wells, hold the Control key and click each well.

With the target wells selected, right-click and select Paste Well.

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Chapter 8 Preparing Plates

CFX Maestro Dx SE pastes the contents of the first well into the selected wells.

Adding a Note to a Well
You can add a descriptive note to a well. You can view the well notes in the Quantification tab in the Data
Analysis window.

To add a note to a well1.

In the plate pane, select the well or wells to which you plan to add a note.

2.

In the View section in the bottom pane, select Well Note.
The Well Note area appears in the right pane.

3.

Type the content for the note in the textbox and press Enter.
The text appears at the bottom of the selected wells.
Tip: If you created a previous well note, you can select it from the dropdown list and apply it to
the selected wells.

Clearing Wells of All Content
You can clear an individual well, a group of wells, or the whole plate of all content. Clearing wells does notremove the fluorescence data collected during the plate read.
Important: Clearing a well permanently removes the content from the well. If you click OK and savethe plate after clearing a well, you cannot undo the clear action. Take care when clearing wells.

To clear wells of all settings
1.

2.

In the Plate Editor, select the well or group of wells in the plate pane:n

To select a single well, click on the well.

n

To select multiple adjacent wells, click a well and drag to the target well.

n

To select multiple nonadjacent wells, hold the Control key and click each well.

n

To select an entire column with the same sample type, click the column number.

n

To select an entire row, click its row number.

In the right pane, click Clear Wells.

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Assigning Optional Parameters to the Plate File

CFX Maestro Dx SE clears the selected wells of all settings.
3.

Do one of the following:n

If you cleared the wells in error, click Undo on the Plate Editor toolbar before you click OK toaccept the changes.
Important: Clicking OK before you click Undo saves the changes and disables Undo on the
Plate Editor toolbar.

n

Click OK to accept the changes and save the plate.

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Chapter 8 Preparing Plates

Changing Experiment Settings
Use the Experiment Settings dialog box to view or change the list of targets, samples, or biologicalgroups, or to set the gene expression analysis sample group to analyze if you assigned biological groups
to wells in the plate.
In the Experiment Settings dialog box, the Targets tab displays a list of target names for each PCR
reaction, such as the target gene or gene sequences of interest.
The Samples and Biological Groups tab displays a list of sample and biological group names that indicate
the source of the target, such as a sample taken at 1 hour (1Hr) or from a specific individual (mouse1).

To change plate settings using the Experiment Settings dialog box
1.

To open the Experiment Settings dialog box, do one of the following:n

In the right pane in the Plate Editor, click Experiment Settings.

n

In the Gene Expression tab in the Data Analysis window, click Experiment Settings.

The Experiment Settings dialog box appears displaying the contents of the Targets tab.

2.

To add a new target, sample, or biological group name, in the appropriate tab type a name in the New
textbox and click Add.

3.

To remove one or more target, sample, or biological group names from the list, in the appropriate tab
select the item’s checkbox in the Select to Remove column and click Remove checkeditem(s).

140 | CFX Maestro Dx SE SoftwareChanging Experiment Settings

4.

CFX Maestro Dx SE excludes the sample type NTC (no template control) from gene expression
analysis.
To include NTC sample types, clear its checkbox in the Exclude the following sample types section.
You can choose to exclude the following sample types by selecting the appropriate checkbox:

5.

n

NRT (no reverse transcriptase)n

Negative Control

nPositive Control

nStandard

In the Targets tab:a.

To select a target as the reference for gene expression data analysis, select it in the Reference
column.

b.

To hide analysis settings that will be applied in the Gene Expression tab in the Analysis Settings
window, clear Show Analysis Settings.
The software hides the following columns:
n

Color

n

Show Chartn

Auto EfficiencynEfficiency (%)

c.

To change the color of the target as it is graphed in the Gene Expression chart, click its cell in the
Color column, select a new color in the Color dialog box that appears, and click OK.

d.

To display the target in the selected color in the Gene Expression chart, select its checkbox in
the Show Chart column.

e.

By default, CFX Maestro Dx SE automatically calculates the relative efficiency for a target if its
data include a standard curve.
To use a previously determined efficiency value, type the value in its cell in the Efficiency (%)
column and press the Enter key. CFX Maestro Dx SE clears the Auto Efficiency checkbox.

6.

In the Samples and Biological Groups tab:a.

To select a sample or biological group as the control sample for gene expression data analysis,
select its checkbox in the Control column.

b.

To assign the control condition to a sample or biological group for a run, click its checkbox in theControl column.

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Chapter 8 Preparing Platesc.

7.

If it is not already selected, click Show Analysis Settings to view or change analysis parametersthat will be applied in the Gene Expression tab. The software hides the Color and Show Chart
columns.

Click OK to save the parameters in the Experiment Settings dialog box and return to the Plate Editor
window.

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Creating Well Groups

Creating Well Groups
Well groups divide a single plate into subsets of wells that can be analyzed independently in the Data
Analysis window. Once well groups are set up, select one in the Data Analysis window to analyze the dataas an independent group. For example, set up well groups to analyze multiple experiments run in one
plate or to analyze each well group with a different standard curve.
Note: The default well group is All Wells.

To create well groups
1.

To open Well Groups Manager, do one of the following:n

In the Plate Editor toolbar, click Well Groups.

n

In the Data Analysis window, click Manage Well Groups.

The Well Groups Manager dialog box appears.

2.

Click Add to create a new group. The dropdown menu displays the group name as Group 1 for the
first group.

3.

Select the wells for the well group in the plate view by clicking and dragging across the group of wells.
Selected wells appear blue in the Manager.

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Chapter 8 Preparing Plates

4.

(Optional) To change the name of the group, select its name in the dropdown menu and type a new
name.

5.

(Optional) To delete a well group, select its name in the dropdown list and click Delete.

6.

Click OK to finish and close the window, or click Cancel to close the window without making changes.

Right-Click Menu Items for the Well Groups Manager Dialog Box
Table 10 lists the menu items available in the Well Groups Manager dialog box when you right-click on
any well.
Table 10. Right-click menu items in the Plate Editor Well Groups Manager dialog box

Item

Function

Copy

Copies the well contents, which can then be pasted intoanother well or wells.

Copy as Image

Copies the well selector view as an image.

Print

Prints the well selector view.

Print Selection

Prints only the selected cells.

Export to Excel

Exports the data to an Excel spreadsheet.

Export to CSVExports the data as a comma-separated document.

Export to Xml

Exports the data as an .xml document.

Export to Html

Exports the data as an .html document.

144 | CFX Maestro Dx SE SoftwareChanging Trace Styles

Changing Trace Styles
During plate setup and while a run is in progress, you can modify the color and style of the amplification
traces. You can then easily view the traces in the real-time status window as the data are being collected.

To change trace styles
1.

Click Trace Styles in the Plate Editor toolbar.
The Trace Styles dialog box appears for the open plate, for example:

2.

To display the trace styles by a specific fluorophore, select it from the Fluorophores dropdown.

3.

To change the trace display:a.

Select the trace type from the Wells dropdown list.

b.

Click its color in the Color column.

c.

In the Color dialog box that appears, choose another color for the trace and click OK.
CFX Maestro Dx SEdisplays the color change for the well type in the grid.

d.

(Optional) Select a symbol for the trace from the Symbols dropdown list.

4.

To quickly change the color set, click the appropriate choice in the Color Quick Set section.

5.

To view well labels in the grid, select the label type in the Well Labels section.

6.

Click OK to save changes or Cancel to cancel changes.

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Chapter 8 Preparing Plates

Viewing, Exporting, and Importing the Plate in Spreadsheet
FormatThe Spreadsheet View/Importer tool displays the contents of a plate in spreadsheet format. The viewer
provides option to view, import, and export well data as described below.

Using the Spreadsheet Viewer to Export and Import Plate Data
From the spreadsheet viewer, you can export the Target Name, Sample Name, Biological Group Name,and Well Notes as a template in a tab-delimited format to an application such as Microsoft Excel. You canalso import that data from a tab-delimited application into a pre-defined plate from an experiment
information file.

To use the Spreadsheet View/Importer tool
1.

Create and save a plate file (see Creating a Plate File Using the Plate Editor).

2.

On the Plate Editor toolbar, click the Spreadsheet View/Importer tab to open the Plate Spreadsheet
View dialog box.

3.

(Optional) Click the Show Biological Set Name and Show Well Note boxes to display those columns
in the Spreadsheet View and in the exported file.

4.

Click the Export Template button to create an empty template in an Excel file (.csv format). The
exported file will display the same layout as your plate.
Tip: Use the plate file name when saving your plate files to easily identify the file.

5.

Populate the Excel file cells with your well content.

146 | CFX Maestro Dx SE SoftwareViewing, Exporting, and Importing the Plate in Spreadsheet Format

Note: You can only edit the contents of any cell in a column that has an asterisk (*) beside thecolumn name (*Target Name, *Sample Name, *Biological Group Name, *Well Note).
Note: You cannot add values to the Standard Curve and Quantity Columns in the exported
Excel file. To modify that data, return to the Plate editor and select Settings > Units in the menubar. After the plate run is completed, the data from these standards appear in the Standard
Curve chart in the Quantification tab in the Data Analysis window with the units you select.
6.

Import the populated Excel file back into the Plate Editor by clicking the Import button. The imported
plate data appears in the Plate Spreadsheet View window.
Important: If you have multiple fluorophores, you will need to perform steps 3-5 for eachfluorophore using the Flours List dropdown menu in the Plate Spreadsheet View.

7.

Click the OK button. The new plate data now appears in the Plate Editor window.

Tip: You can view menu items available in the Spreadsheet View/Importer tool when you any rightclick on any well in the tool or on any of the table headers of the Plate Spreadsheet view.

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Chapter 8 Preparing Plates

Creating a Plate Layout Using the Plate Setup Wizard
You can use the Setup Wizard to enter the plate layout information that is needed for normalized gene
expression analysis, including:nTarget namesnSample namesnLocation of targets and sample on the platenReference gene(s)nControl sample

You can use the Setup Wizard before, during, or after a run.

Using the Plate Setup Wizard
This section explains how to create a plate layout using the plate Setup Wizard. To view the contents ofeach well in the plate more easily, click Zoom plate at the top of the Setup Wizard.
Important: Returning to the Auto layout tab while on any other tab in the Setup Wizard resets theplate layout. Take care when selecting this tab.
Tip: You can reset the layout by selecting Tools > Clear Plate in the Setup Wizard.

To use the plate Setup Wizard
1.

Open the Plate Editor.

2.

To open the Setup Wizard, do one of the following:nChoose Editing Tools > Setup Wizard.

n

Click Setup Wizard on the Plate Editor toolbar.

The Setup Wizard appears displaying the Auto layout tab.

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Creating a Plate Layout Using the Plate Setup Wizard

3.

In the Auto layout tab, do the following:a.

Click a well in the grid and drag across and down to specify the area on the plate in which youplan to load sample.

b.

Enter the number of targets and samples to load.
Tip: The number of targets and samples must equal the number of selected cells. If thenumbers entered do not fit in the area selected, modify the numbers or plate selection area.
The orientation of items on the plate and their grouping can be specified.

c.

(Optional) Change the plate orientation. For example, you can set targets in columns andsamples in rows, or group by samples.

d.

Click Next to proceed to the Target names tab.

Note: If your plate layout does not have a regular pattern, use the Target names tab to manually
position your targets or the Sample names tab to manually position your samples on the plate.
Click and drag to select multiple wells.
4.

In the Target names tab, define target names for the target groups:a.

b.

Do one of the following:n

To rename targets by group, set Select by to Target.

n

To rename targets by well, set Select by to Well.

Select a target group or well in the grid and type a name in the Target name dropdown list.

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Chapter 8 Preparing Plates

Tip: Press Tab to select the next group or well to the right or Enter to select the next groupor well below. Alternatively, on the Target name and Sample name tabs, hold the Control
key and click a well to select multiple wells that are not adjacent.
c.

Click Next to proceed to the Sample names tab.

5.

In the Sample names tab, define sample names for the sample groups.

6.

Click Next to proceed to the Reference targets tab.

7.

In the Reference targets tab, select one or more targets to use as references for normalized gene
expression and click Next to proceed to the Control sample tab.

8.

In the Control sample tab, select one sample to use as a control for relative gene expression
calculations.

9.

Click OK to save the plate layout and return to the Plate Editor, in which you can further define plateparameters. See Assigning Optional Parameters to the Plate File on page 129 for more information.
Alternatively, click Previous to return to a previous tab to make any changes.
Note: Returning to the Auto layout tab automatically resets the plate. Take care when clicking
Previous.

150 | CFX Maestro Dx SE SoftwareChapter 9 Running Experiments
This chapter explains how to run custom (user-defined) or PrimePCR assay experiments using
CFX Maestro Dx Software, Security Edition.
A run data file contains the protocol and plate information for the run. The file also contains the data fromthe analyses that CFX Maestro Dx SE performs after the run completes.
CFX Maestro Dx SE makes it easy to set up and run user-defined or PrimePCR experiments. The Run
Setup window guides you through the common steps of setting up an experiment, leading you to the Start
Run dialog box, from which you start the run.

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Chapter 9 Running Experiments

The Run Setup Window
The Run Setup window provides quick access to the files and settings needed to set up and run anexperiment. When you choose to run a user-defined experiment, the Run Setup window opens displaying
the Protocol tab. When you choose to run a PrimePCR experiment, the Run Setup window opens
displaying the Start run tab.
Tip: See Performing PrimePCR Experiments on page 168 for information about PrimePCR; seeStart Run Tab on page 158 for information about the Start Run tab.

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The Run Setup Window

LEGEND
1.

Tabs guide you through setting up and running an experiment:n Protocol tab — select an existing protocol to run or edit, or to create a newprotocol in the Protocol Editor.
n Plate tab — select an existing plate to run or edit, or to create a new plate in the
Plate Editor.
n Start Run tab — view the experiment settings, select one or more instrument
blocks, and begin the run.

2.

The main window displays the options for each tab as you apply them.

3.

Navigation buttons lead you to the Start Run tab.

Accessing the Run Setup Window
To access the Run Setup windowu

Do one of the following:n

In the Run setup tab in the Startup Wizard, click either User-defined or PrimePCR.

n

In the Home window, click either User-defined Run Setup or PrimePCR Run Setup on the
toolbar.

n

In the Home window, select either Run > User-defined Run or Run > PrimePCR Run.

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Chapter 9 Running Experiments

Protocol Tab
The Protocol tab displays a preview of the protocol file that you plan to run. A protocol file contains the
instructions for the instrument temperature steps as well as instrument options that control the ramp rate,
sample volume, and lid temperature.

By default, the software displays the protocol defined in the File Selection for Run Setup section in theFiles tab in the User > User Preferences dialog box. You can change the default protocol in the User
Preferences dialog box. See Changing the Default File Settings on page 79 for more information.
In the Protocol tab, you cannCreate a new protocol to runnSelect an existing protocol to run or edit

For more information about creating and modifying protocols, see Chapter 7, Creating Protocols.

To create a new protocol
1.

On the Protocol tab, click Create New.
The Protocol Editor appears.

2.

Use the Protocol Editor to create a new protocol.

154 | CFX Maestro Dx SE SoftwareProtocol Tab

3.

Click OK to save the protocol and return to the Protocol tab in Run Setup.

4.

View the details of the protocol and do one of the following:n

If the details are correct, click Next to proceed to the Plate tab.

n

If the details are incorrect, click Edit Selected to return to the Protocol Editor window. Revise theprotocol, save changes, and then click Next on the Protocol tab to proceed to the Plate tab.

To select an existing protocol
1.

On the Protocol tab, do one of the following:n

Click Select Existing and navigate to an existing protocol.

nClick Express Load and select a protocol from the dropdown list of protocols.
Tip: You can add protocols to or remove them from the Express Load dropdown list. See
Adding and Removing Express Load Protocols that follows for more information.

2.

View the details of the protocol and do one of the following:n

If the details are correct, click Next to proceed to the Plate tab.

n

If the details are incorrect, click Edit Selected to open the Protocol Editor. Revise the protocol,
save changes, and then click Next on the Protocol tab to proceed to the Plate tab.

Adding and Removing Express Load Protocols
You can modify the contents of the Express Load dropdown list that appears in the Protocol Editor. The
protocols in this list are saved in the following folder:
c:\Users\Public\Public Documents\Bio-Rad\CFX_MDx\Users\<user_name>\ExpressLoad\To modify the Express Load list of protocols
1.

Navigate to and open the ExpressLoad folder.

2.

Review the protocol files (.pcrl) in the folder.

3.

Do either of the following:nDelete protocols from the folder to remove them from the dropdown list.

n

Copy protocols into the folder to add them to the dropdown list.

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Chapter 9 Running Experiments

Plate Tab
Note: If the protocol selected in the Protocol tab does not include a plate read step for real-time PCR
analysis, the Plate tab is hidden. To view the Plate tab, add at least one plate read to the protocol.
The Plate tab displays a preview of the plate file you plan to load. In a real-time PCR run, the plate filecontains a description of the contents of each well including its fluorophores, the scan mode, and the platetype. CFX Maestro Dx SE uses these descriptions for data collection and analysis.

By default, the software displays the plate defined in the File Selection for Run Setup section in the Filestab in the User > User Preferences dialog box. You can change the default plate in the User Preferences
dialog box. See Changing the Default File Settings on page 79 for more information.
In the Plate tab, you cannCreate a new plate to loadnSelect an existing plate to load or edit

For more information about creating and modifying plates, see Chapter 8, Preparing Plates.

156 | CFX Maestro Dx SE SoftwarePlate Tab

To create a new plate1.

On the Plate tab, click Create New.
The Plate Editor appears.

2.

Use the Plate Editor to create a new plate.

3.

Click OK to save the plate and return to the Plate tab in Run Setup.

4.

View the details of the plate and do one of the following:n

If the details are correct, click Next to proceed to the Start Run tab.

n

If the details are incorrect, click Edit Selected to return to the Plate Editor. Revise the plate file,save changes, and then click Next on the Plate tab to proceed to the Start Run tab.

To select an existing plate file1.

On the Plate tab, do one of the following:n

Click Select Existing and navigate to an existing plate file.

n

Click Express Load and select a plate file from the dropdown list.
Tip: You can add plates to or remove them from the Express Load dropdown list. See
Adding and Removing Express Load Plate Files that follows for more information.

2.

View the details of the plate and do one of the following:n

If the details are correct, click Next to proceed to the Start Run tab.

n

If the details are incorrect, click Edit Selected to open the Plate Editor window. Revise the plate file,save changes, and then click Next to proceed to the Start Run tab.

Adding and Removing Express Load Plate Files
You can modify the contents of the Express Load dropdown list that appears in the Plate Editor. The
plates that appear in this list are saved in the following folder:
c:\Users\Public\Documents\Bio-Rad\CFX_MDx\Users\<user_name>\ExpressLoad\To modify the Express Load list of plate files
1.

Navigate to and open the ExpressLoad folder.

2.

Review the plate files (.pltd) in the folder.

3.

Do one of the following:nDelete plate files from the folder to remove them from the dropdown list.

n

Copy plate files into the folder to add them to the dropdown list.

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Chapter 9 Running Experiments

Start Run Tab
The Start Run tab displays information about the experiment to run. It also displays the connected
instrument block or blocks on which you can run the experiment.

In the Start Run tab you can do the following:nView detailed run information, including the selected protocol file, plate file, and scan mode.

n

Add notes about the run.

nView details about all connected instruments, including their run status (running or idle), sample
volume in µl, lid temperature, emulation mode, and ID or bar code if available.
Note: You can modify the columns that appear in the Start Run on Selected Blocks table. See
Modifying Details in the Selected Blocks Table on page 159 for information.

nSelect the block or blocks on which to perform the run.

nRemotely open or close the lid of each selected instrument.

nStart the run.

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Start Run Tab

Modifying Details in the Selected Blocks Table
You can modify the columns that appear in the Start Run on Selected Block(s) table. You can also modify
the default sample volume and lid temperature values in the table. The setting changes are applied to therun to be performed.

To add columns in the Start Run on Selected Blocks tableuRight-click the table and select an option in the menu that appears.

To remove columns in the Start Run on Selected Blocks tableuRight-click the table and clear the option in the menu that appears.

To edit sample volume or lid temperature values for a blocku

Select the sample volume or lid temperature cell for the target block and type a new value into thecell.

To add a run ID or bar code for a blocku

Select the ID/Bar Code cell for the target block and type an ID or scan the block with a bar code
reader.

Running an Experiment
Important: Before running an experiment, make sure your computer's anti-virus software will notinitiate a scan during the run. See Installing CFX Maestro Dx SE Software on page 31 and yoursystem administrator for more information.

To run an experiment
1.

In the Start Run tab, verify the plate and protocol details in the Run Information section.

2.

(Optional) Add notes about the run or experiment in the Notes text box.

3.

Select the checkbox of one or more blocks on which to perform the run.
Tip: To run the experiment on all blocks, select Select All Blocks located below the Selected
Blocks table.

4.

(Optional) Click Flash Block Indicator to flash the indicator LED on the selected instrument blocks.

5.

Insert experiment plates into the block:a.

Click Open Lid. The motorized lid of each selected block opens.

b.

Insert an experiment plate into each selected block.

c.

Click Close Lid.

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Chapter 9 Running Experiments

Tip: On CFX Opus Dx systems, tap Open Lid or Close Lid on the Home screen.
6.

Click Open Lid and Close Lid to open and close the motorized lid of each selected instrument block.

7.

View the details of the run and do one of the following:n

If the details are correct, click Start Run.

n

If the details are incorrect:oCorrect the details in the Selected Blocks table and click Start Run.

oReturn to the correct tab and make the appropriate changes, save the changes and thenclick Next to return to the Start Run tab and start the run.

To start a new run from a previous runu

Do one of the following:nSelect File > Repeat a Run in the main software menu bar; navigate to and double-click the rundata file that you want to repeat.

nSelect the Repeat Run tab in the Startup Wizard and double-click the run data file of the run you
want to repeat.
Optionally, on the Repeat Run tab you can click Browse and navigate to and double-click the rundata file that you want to repeat.

160 | CFX Maestro Dx SE Software

Run Details Dialog Box

Run Details Dialog Box
When you click Start Run, CFX Maestro Dx SE prompts you to save the data file (.pcrd), starts the run,and opens the Run Details dialog box. The Run Details dialog box contains three status tabs:n

Run Status — use this tab to view the current status of the protocol, open or close the lid, pause arun, add repeats, skip steps, or stop the run.

n

Real-time Status — use this tab to view the real-time PCR fluorescence data as they are collected.

nTime Status — use this tab to view a full-screen countdown timer for the protocol.

These tabs are explained in detail in the sections that follow.

Run Status Tab
The Run Status tab displays the current status of a run in progress. In this view you can also control the lid
and change the run in progress.

LEGEND
1.
2.

Run Status pane — displays the current progress of the protocol.
Run Status controls — enable you to operate the instrument or to interrupt thecurrent protocol.

3.

Run Information pane — displays run details.

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Chapter 9 Running Experiments

Run Status Commands
Use the commands in the Run Status tab to either operate the instrument from the software or change a
run that is in progress.
Note: Making changes to the protocol during the run, such as adding repeats, does not change theprotocol file associated with the run. These actions are recorded in the Run Log.

— opens the motorized lid on selected instruments.
Important: Opening the lid during a run pauses the run during the current step and might alter
the data. Run Status Commands on page 162.

— closes the motorized lid on selected instruments.

— adds more repeats to the current GOTO step in the protocol. This option is available only when a
GOTO step is running.
Note: You can add additional repeats while in a GOTO cycle when the protocol is in progress.
However, CFX Maestro Dx SE recognizes the most recent change in the number of repeats. For
example, if you add 10 additional repeats while in a GOTO cycle, the software will change thetotal number to n + 10. If you then add an additional five (5) repeats while in the same cycle, CFX
Maestro will change the total number of repeats to n + 5. The first change (10 repeats) is ignored.
To ensure the software performs the target number of repeats, enter the total number (in thiscase, 15 repeats).

— skips the current step in the protocol.
Note: If you initiate a skip during a GOTO step, the system skips to the next cycle in the GOTO
loop. If the GOTO step’s last cycle was in progress at the time of the skip, the system skips to thenext step.

— flashes the LED on the selected instrument to identify the selected blocks.

— pauses the protocol.
Note: This action is recorded in the Run Log.

— resumes a paused protocol.

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Run Details Dialog Box— stops the run before the protocols ends.
Note: Stopping a run before the protocol ends might alter your data.

Opening the Instrument Lid During a PCR Run
If the lid of any instrument is opened during a PCR run, the CFX Maestro Dx SE will display the following
confirmation dialog:

While the dialog appears, the instruments continues to run the protocol. The OK button pauses the run and the
instrument lid unclamps and opens. The Cancel button dismisses the dialog and resumes the run.

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Real-time Status Tab
The Real-time Status tab displays real-time PCR data collected at each cycle during the run after the firsttwo plate reads.

LEGEND
1.
2.

Amplification trace pane — displays real-time amplification data during the run.
Well group identifier — if well groups were identified in the plate setup, users canselect a specific well group to view its traces, wells, and tabular information.
Step number identifier —if the protocol collects data at more than one step (forexample during amplification and melt curve), users can select a specific step andview the traces collected at that step.

3.

Well selector pane — displays the active, inactive, and empty wells in the plate.

4.

Plate setup table pane — displays the plate setup in tabular format.

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Run Details Dialog Box

5.

Run details pane — displays the real-time status of the run including:n Current stepn Current repeatn Current temperaturen Time remainingn Sample temperature
n Lid temperature

6.

Plate Setup — opens the Plate Setup dialog box, in which users can modify thecurrent plate setup during a run.

In the Real-time Status tab you cannShow or hide real-time traces by selecting them in the well selector pane or the plate setup tablenView single or groups of traces by selecting them in the well-group dropdownnEdit the plate or replace the plate filen

Apply a PrimePCR file to the run.

Showing or Hiding Real-Time Traces
By default, all filled wells are active and appear in the plate setup table. Active wells appear blue in thewell selector pane. Hidden wells appear light gray, and unused wells appear dark gray in the well selector
pane.
You can hide traces from active wells during the run. CFX Maestro Dx SE continues to collect data for all
wells; when you hide wells, their data do not appear in the plate setup table.

To hide real-time tracesu

In the well selector pane, click the active (blue) wells that you want to hide.

To show real-time tracesu

In the well selector pane, click the hidden (light gray) wells that you want to display.

For more information about the well selector, see Well Selector on page 185.

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Editing a Plate Setup
To edit a plate setupu

Click Plate Setup and then select View/Edit Plate.
The Plate Editor window appears, in which you can edit the plate while the run is in progress. For
more information about editing plates, see Chapter 8, Preparing Plates.
Note: You can also edit the trace styles from the Plate Editor window. Changes appear in theamplification trace plot in the Real-time Status tab.

Replacing a Plate File
Tip: Replacing a plate file is especially useful if you start a run with a Quick Plate file in the
ExpressLoad folder.

To replace a plate fileu

Click Plate Setup and then select one of the following options:nReplace Plate file — select the new plate file from the list in the browser windownApply PrimePCR file — search for a run file from which the plate layout will be obtained using
Smart search or click Browse to find a file that you downloaded from the Bio-Rad website andthat is not located in the PrimePCR folder

Note: CFX Maestro Dx SE checks the scan mode and plate size for the plate file. These must be
the same as the run settings with which the run was started.

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Run Details Dialog Box

Time Status Tab
The Time Status tab displays the time remaining to complete the current run.

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Performing PrimePCR Experiments
PrimePCR experiments use pathway or disease-specific assays that Bio-Rad has wet-lab validated andoptimized and are available in the following formats:n

Preplated panels — plates containing assays that are specific for a biological pathway or disease;
they include PrimePCR controls and reference genes.

nCustom configured plates — plates that can be set up in a user-defined layout with the option tochoose assays for targets of interest, controls, and references.

n

Individual assays — tubes that contain individual primer sets for use in real-time reactions.

To reduce the overall run time, you can remove the melt step in the protocol. Bio-Rad strongly
recommends that you do not make any other modifications to a PrimePCR run protocol. The default
protocol is the one that was used for assay validation. Any deviation from this may affect the results.
Protocol changes are noted in the Run Information tab of the resultant data file and in any reports that are
created.

To start a PrimePCR runu

To start a PrimePCR run, do any of the following:n

In the Startup Wizard, select PrimePCR on the Run setup tab and then select the appropriate
chemistry (SYBR® or Probe).

nSelect a PrimePCR run from the Recent Runs list on the Repeat run tab in the Startup Wizard.

nSelect File > Open > PrimePCR Run File on the Home window.

nDrag and drop a PrimePCR run file onto the Home window.

After you select a PrimePCR run, the Run Setup window opens on the Start Run tab with the default
PrimePCR plate layout loaded based on the selected instrument.

To remove the melt step in the protocoluOn the Protocol tab, clear the box adjacent to Include Melt Step.

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Performing PrimePCR Experiments

To import target information for PrimePCR plates into a plate layout
1.

Do one of the following:n

In the Real-time Status tab in the Run Details dialog box, select Plate Setup > Apply PrimePCR
File.

n

In the Data Analysis window, select Plate Setup > Apply PrimePCR File.

2.

In the PrimePCR run file dialog box, click Browse to navigate to the appropriate PrimePCR file (.csv).

3.

Select the target PrimePCR file and click Open.

CFX Opus Dx system imports the target information into your plate layout.

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Chapter 9 Running Experiments

Transferring Stand-Alone Data for Analysis
Important: When you transfer data files from the CFX Opus Dx system to CFX Maestro Dx SE, allfiles saved on the system are transferred. Ensure you have enough disk space for the data to transfer
safely.
When the run is complete, CFX Maestro Dx SE analyzes the fluorescence data. If the run is performed instand-alone mode and saved on the CFX Opus Dx system itself, the data need to be transferred to the
CFX Maestro Dx SE computer for analysis.
The CFX Opus Dx system can store up to 100 real-time PCR runs. After the run completes, you cantransfer stand-alone data files to the CFX Maestro Dx SE computer through email, USB drive, or throughthe software itself.
This section explains how to transfer stand-alone data files to the CFX Maestro Dx SE computer.

Transferring Data Through EmailTo email a data file at the end of a run
1.

Set up email notifications for the instrument.
See Setting Up Email Notification on page 76 or the CFX Opus Dx Real-Time PCR System Operation
Manual.

2.

When you set up email notifications, ensure that Attach Data File is selected.
The run data are emailed as a .pcrd file.

Transferring Data from CFX Opus Dx Real-Time PCR Systems
Using the File Browser feature on the CFX Opus Dx system, you can transfer data files to an attached
USB drive or to a shared network folder. You can also transfer CFX Maestro Dx SE protocol files from a
USB drive or shared network drive to your folder or the Public folder on the CFX Opus Dx system and run
them on the CFX Opus Dx system.
Tip: This section explains how to transfer data. For information about setting up the Ethernet, see the
CFX Opus Dx Real-Time PCR System Operation Manual available on the CFX Maestro Dx SE Helpmenu.

Copying Files1.

On the CFX Opus Dx system’s Home screen, tap Files to view the File Browser screen.

2.

On the File Browser screen, navigate to the file you want to copy, then tap the file to view the filedetails pane.

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Transferring Stand-Alone Data for Analysis

3.

In the file details pane, tap Options and then tap Copy.

The Select Location dialog box appears.
4.

In the Select Location dialog box, do one of the following:nNavigate to an existing folder.

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Chapter 9 Running ExperimentsnNavigate to the location to create a folder in which to save the file, then tap Create Folder(

5.

) to create a new folder in that location.

Tap Select to copy the file to the selected location or Cancel to return to the File Browser screen.
Note: If a file with the same name exists at the selected location, a message box appears. Tap
Yes to overwrite the existing file or No to return to the File Browser screen.
The CFX Opus Dx system displays a confirmation message when the file is successfully copied.

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Transferring Data through CFX Maestro Dx Software, Security
EditionTo transfer data through CFX Maestro Dx SE
1.

In the Detected Instruments pane on the Home window, right-click the target instrument and selectRetrieve Data Files.
CFX Maestro Dx SE displays the Browse For Folder dialog box.

2.

In the Browse For Folder dialog box, browse to the location in which you plan to save the data files
and click OK.
The transfer process creates a folder labeled Real-Time Data in the selected location. The run data
are saved into the Real-Time Data folder as separate .zpcr files.

Transferring Data Using a USB Drive
If you insert a USB drive into the USB port on the instrument, the data file is automatically saved to theroot directory of the USB drive when the run is complete. You can also locate previously saved data files
and save them onto an attached USB drive.

To transfer data files onto a USB drive on CFX Opus Dx systemsu

In the Select Location dialog box, tap USB and navigate to the target folder in which to copy the file orCancel to return to the File Browser screen.
Note: If a file with the same name exists at the selected location, a dialog appears. Tap Yes tooverwrite the existing file or No to return to the File Browser screen.

The CFX Opus Dx system displays a confirmation message when the file is successfully copied.

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Chapter 9 Running Experiments

Transferring Data through a Shared Network Drive Using CFX
Opus Dx Real-Time PCR Systems
Tip: You can transfer data to and from a shared network drive only through CFX Opus Dx systems.
CFX Opus Dx systems enable you to connect to a shared network drive using Ethernet. With a successfulconnection, you can transfer data files to and from a folder on the shared network drive.

To transfer data to and from a shared network driveu

In the Select Location dialog box, tap Network and navigate to the target folder in which to copy thefile or Cancel to return to the File Browser screen.
Note: If a file with the same name exists at the selected location, a dialog appears. Tap Yes tooverwrite the existing file or No to return to the File Browser screen.

The CFX Opus Dx system displays a confirmation message when the file is successfully copied.

Creating a Data FileTo analyze data transferred from the instrument to the CFX Maestro Dx SE computer, the compresseddata file (.zpcr file) must be converted to a data file (.pcrd file). CFX Maestro Dx SE converts the .zpcr file
to a .pcrd file and then selects a plate file that has the same the scan mode and plate size and applies it to
the .pcrd file.

To create a data file from a stand-alone data file1.

In CFX Maestro Dx SE do one of the following:n

Locate the target .zpcr file and drag it onto the CFX Maestro Dx SE Home window.

nSelect File > Open > Stand-alone Run and navigate to and select the target file.

CFX Maestro Dx SE displays the Save As dialog box.
2.

Navigate to the folder in which you plan to save the .pcrd file and click Save.
After you save the .pcrd file, CFX Maestro Dx SE opens the Data Analysis window and displays theresulting data.

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CFX Maestro Dx Software, Security Edition processes real-time PCR data automatically at the end ofeach run and opens the Data Analysis window to display these data (the .pcrd file).
nDrag a data file (.pcrd extension) onto the Home window and release itn

Select File > Open > Data File in the Home window and browse to the target .pcrd filenSelect File > Recent Data Files in the Home window to select from a list of the ten most recently
opened data filesnChoose the Analyze tab in the Startup Wizard and either select from Recent Files or click Browse to
locate the data file

Data Analysis Window
The Data Analysis window displays multiple tabs, each tab showing the analyzed data for a specificanalysis method or run-specific information. Tabs appear only if the data collected in the run are available
for that type of analysis.

Tip: To choose the tabs to display, select them from the dropdown menu View in the Data Analysis
window. To return to the original tab layout, select Settings > Restore Default Window Layout.

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Chapter 10 Data Analysis Overview

Data Analysis Toolbar
The toolbar in the Data Analysis window provides quick access to important data analysis functions.

Table 11 lists the functions of buttons in the toolbar.
Table 11. Toolbar in the Data Analysis window

ButtonName

Function

Plate Setup

View/Edit plate — opens the Plate Editor to viewand edit the contents of the wells.
Replace Plate — selects a plate file to replace theplate layout.
Apply PrimePCR file — selects a run file toreplace the plate layout for a PrimePCR run.

Manage Well

Opens the Well Groups Manager window toGroupscreate, edit, and delete well groups.

Well Group

Selects an existing well group name from thedropdown menu. The default selection is All
Wells. This button appears only when well groups
are created.

Analysis Mode

Analyzes the data in either Fluorophore or Target
mode.

Help

Opens the software Help from which you can findonline help and a digital copy of this manual in
Acrobat PDF format.

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Data Analysis Menu Bar
Table 12 lists the menu bar items in the Data Analysis window.
Table 12. Data Analysis window menu bar items

Menu Item

CommandFunction

File

Save

Saves the file.

Save As

Saves the file with a new name.

File Passwords

Enables users to set file save and open
passwords.

Sign

Enables users to sign the data file.

Repeat Run

Extracts the protocol and plate file from thecurrent run to rerun it.

View

Close

Closes the Data Analysis window.

Run Log

Opens a Run Log window to view the run log ofthe current data file.

Audit Trail

Opens the audit trail for the file.

Quantification, Melt Curve, Gene

Displays the analyzed data in selected tabs in

Expression, End Point, Custom

the Data Analysis window. At least one tab

Data View, QC, Run Informationmust be selected.

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Table 12. Data Analysis window menu bar items, continued

Menu Item

CommandFunction

Settings

Cq Determination Mode

Enables you to select either Regression orSingle Threshold mode to determine how Cq
values are calculated for each trace.

Baseline Setting

Enables you to select the Baseline Subtraction
method for the selected well groups.

Analysis Mode

Enables you to analyze data by Fluorophore or
by Target.

Cycles to Analyze

Enables you to select the cycles to be analyze.

Baseline Threshold

Opens the Baseline Threshold window toadjust the baseline or the threshold.

Trace Styles

Opens the Trace Styles window.

Plate Setup

Opens the Plate Editor to view and edit theplate; replace the current plate with one from auser-defined plate file or a PrimePCR run file.

Include All Excluded Wells

Includes all excluded wells in the analysis.

Mouse Highlighting

Turns on or off the simultaneous highlighting ofdata with the mouse pointer.
Tip: If Mouse Highlighting is turned off,press the Control key to temporarily turnon highlighting.

Restore Default Window Layout

Restores the arrangement of windows to thedefault setting.

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Data Analysis Window

Table 12. Data Analysis window menu bar items, continued

Menu Item

CommandFunction

ExportExport All Data Sheets

Enables you to select whether to export all the
spreadsheet views from every tab to a .csv,.txt. Excel, or .xml file.

Export RDML File

Enables you to select either version 1.1 or 1.0
of RDML in which to export the file.

Custom Export

Opens the Custom Export window in which thefields to be exported and the file format can be
specified.

Export to LIMS Folder

Opens a window to save data in apredetermined format to the LIMS folder.

Manual Export

Opens a window to identify the location to savedata from all spreadsheet views to Excel files
structured specifically for use by Seegene, Inc.
and Bio-Rad Laboratories.
Tip: You can also automatically start the
Seegene Viewer upon export. See Tools
Menu Commands on page 63 for more
information.

Tools

Reports

Opens the Report for this data file.

Well Group Reports

Opens the Well Group Report window to
generate reports for specified well groups.

Import Fluorophore Calibration

Select a calibration file to apply to the current
data file.

qbase+

Launches qbase+ v2.5 directly from current.pcrd file if it is installed.

Generate LIMS PLRN file

Saves the data file as a LIMS-formatted .plrn
file.

Tab Details
Each tab in the Data Analysis window displays data in charts and spreadsheets for a specific analysis
method and includes a well selector to select the data you want to show. When it opens, the Data Analysis

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Chapter 10 Data Analysis Overview

displays the Quantification tab by default. You can use the Amplification chart data in the Quantification
tab to determine the appropriate analysis settings for the run.

Note: The software links the data in the panes of each Data Analysis tab. For example, highlighting awell by placing the mouse pointer over the well in the well selector view highlights the data in all theother panes.

Step Number Selector
The CFX Opus Dx systems can acquire fluorescence data at multiple protocol steps; the software
maintains the data acquired at each step independently. CFX Maestro Dx SE displays the Step Number
selector below the Standard Curve chart on the Quantification tab. When a protocol contains at least one
data collection step, CFX Maestro Dx SE displays the data from the first collection step.
If the protocol contains more than one collection step, you can select another step from the dropdown list.
For example:

When you select a step, the software applies that selection to all the data that are shown in the Data
Analysis window.

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Data Analysis Window

Viewing Well Groups in Data Analysis
Wells in the plate can be grouped into subsets for independent analysis using well groups. When you
create well groups, their group names appear in the Data Analysis window Well Groups dropdown list on
the toolbar.
If you created well groups, the software displays the default well group All Wells when you open the Data
Analysis window, displaying the data in all wells with content in the charts and spreadsheets. Only thewells in that well group loaded with content appear in the well selector, and only data for those wells areincluded in the data analysis calculations.
Tip: To create, edit, and delete well groups, click Manage Well Groups in the toolbar.
Note: If you did not create well groups, the Well Groups dropdown list does not appear in the toolbar.

Changing Well Contents after a Run
During data analysis, changing the way the data are displayed by changing the contents of the wells in the
Plate Editor never changes the fluorescence data that were collected from each well during the run. Afterthe module collects fluorescence data, you cannot delete those data but you can choose to remove datafrom view and analysis.

To change the content of wells after a runu

In the Data Analysis window, click Plate Setup and select one of the following options:nEdit/View Plate — opens the Plate Editor, in which you can make manual changes to the layout.

n

Replace Plate file — opens the Select Plate browser, in which you can navigate to a previously
saved plate file with which to replace the current plate layout.

n

Apply PrimePCR file — opens the Select PrimePCR file dialog box, in which you can navigate
to a PrimePCR run file and apply it to the plate layout.

Tip: You can add or edit information about the contents of the well before a run, during a run, or after
a PCR run completes. You must assign the scan mode and plate size before the run. These
parameters cannot change after the run.

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Chapter 10 Data Analysis Overview

Data Analysis Settings
The Amplification chart data in the Quantification tab show the relative fluorescence unit (RFU) for eachwell at every cycle. Each trace in the chart represents data from a single fluorophore in one well. These
data are used to determine Cq values for each well on a per fluorophore basis. The software uses one oftwo modes to determine Cq values:nRegression — applies a multivariable, nonlinear regression model to individual well traces and thenuses this model to compute an optimal Cq value.

nSingle Threshold — uses a single threshold value to calculate the Cq value based on the thresholdcrossing point of individual fluorescence traces.

Select Settings > Cq Determination Mode to choose the Cq determination mode.

Adjusting the Threshold
In Single Threshold mode, you can adjust the threshold for a fluorophore by clicking on the threshold linein the Amplification chart and moving the mouse pointer vertically. Alternatively, you can specify an exact
crossing threshold for the selected fluorophore.

Baseline Settings
The software automatically sets the baseline individually for each well. The baseline setting determines
the method of baseline subtraction for all fluorescence traces. The software provides three baseline
subtraction options:n

No Baseline Subtraction — displays the data as relative fluorescence traces. Some analysis is notpossible in this analysis mode and therefore the software does not display the Gene Expression, End
Point, and Allelic Discrimination tabs.

nBaseline Subtracted — displays the data as baseline subtracted traces for each fluorophore in awell. The software must baseline subtract the data to determine quantification cycles, construct
standard curves, and determine the concentration of unknown samples. To generate a baseline
subtracted trace, the software fits the best straight line through the recorded fluorescence of eachwell during the baseline cycles and then subtracts the best fit data from the background subtracted
data at each cycle.

nBaseline Subtracted Curve Fit — displays the data as baseline subtracted traces and the softwaresmooths the baseline subtracted curve using a centered mean filter. This process is performed sothat each Cq is left invariant.

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Data Analysis Settings

In addition to these options, you can also select Apply Fluorescent Drift Correction. For wells that haveabnormally drifting RFU values during the initial few cycles of a run, the software derives an estimatedbaseline from adjacent wells for which a horizontal baseline was successfully generated.

To change the baseline subtraction settinguSelect Settings > Baseline Setting.

Analysis Mode
Data can be grouped and analyzed by either fluorophore or target name. When grouped by fluorophore, data
traces are displayed by fluorophore as indicated in the plate setup for that run. Individual fluorophore data
appear in the amplification and standard curve chart (if available) when the appropriate fluorophore selector
checkboxes, located below the amplification chart, are selected.

When grouped by target, data traces are displayed by target name as entered in the plate setup for thatrun.

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Chapter 10 Data Analysis Overview

To choose a data analysis modeu

Do one of the following:nSelect Settings > Analysis Mode.

nChoose a mode from the Analysis Mode dropdown menu in the toolbar.

Cycles to Analyze
You can restrict the number of cycles to analyze. You can also analyze data from a specific set of cycles.
The maximum number cycles you can analyze is 50.
Note: Removing cycles from the beginning of a run can have a significant impact on baselining.

To restrict data analysis to a specific range of cycles
1.

Select Settings > Cycles to Analyze.
The Cycles to Analyze dialog box appears.

2.

Enter the starting and ending cycle values and click OK.

Click Restore Defaults in the Cycles to Analyze dialog box to return to the cycles originally used foranalysis.

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Well Selector

Well Selector
Use the Well Selector to display or hide the well data in the charts or spreadsheets throughout the Data
Analysis window. Only wells loaded with sample can be selected in the well selector. The software colors
the wells in the Well Selector:nBlue — indicates selected wells. The data from selected wells appear in the Data Analysis window.

nLight gray — indicates unselected wells. The data from unselected wells do not appear in the Data
Analysis window.

nDark gray — indicates empty wells.

To display or hide well datau

In the well selector, do any of the following:n

To hide one well, click the individual well. To display that well, click the well again.

n

To hide multiple wells, drag across the wells you want to select. To display those wells, dragacross the wells again.

n

Click the top left corner of the plate to hide all the wells. Click the top left corner again to display
all wells.

nClick the start of a column or row to hide those wells. Click the column or row again to display thewells.

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Well Selector Right-Click Menu Items
Table 13 lists the right-click options available in the well selector view.
Table 13. Right-click menu items in the well selector view

Item

Function

Well XX

Displays only this well, removes this well from view, set color for thiswell, or excludes this well from analysis.

Selected Wells (right-click and

Displays only these wells, removes these wells from view, sets color for

drag)these wells, or excludes these wells from analysis.

Copy

Copies the content of the well to a clipboard, including Sample Typeand optional Replicate #.

Copy as Image

Copies the well selector view as an image.

Print

Prints the well selector view.

Print Selection

Prints the current selection.

Export to Excel

Exports the data to an Excel spreadsheet.

Export to CSVExports the data as a .csv document.

Export to Xml

Exports the data as an .xml document.

Well Labels

Changes the well labels to Sample Type, Target Name, or Sample
Name.

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Well Selector

Temporarily Excluding Wells from Analysis
To exclude wells from data analysis temporarily
1.

Right-click the well in the well selector, on a fluorescence trace, or on a point plotted on the standard
curve. To exclude multiple wells, right-click and drag to highlight multiple wells, traces, or points.

2.

From the right-click menu, choose the appropriate option:n

Well > Exclude Welln

Selected Wells > Exclude from AnalysisnSelected Traces > Exclude these wells from Analysis

Alternatively, to permanently remove wells from analysis, clear the contents from wells in the Plate
Editor by clicking the Clear Wells button.
Important: You must reenter any well content that is cleared.

To include an excluded wellu

Right-click the appropriate well in the well selector and select Well > Include Well in Analysis.

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Chapter 10 Data Analysis Overview

ChartsEach chart in the Data Analysis window displays the data in a different graph and includes options foradjusting and exporting the data or chart graphics.

Chart Tools
Table 14 lists the right-click options available in most charts.
Table 14. Right-click menu items common to most charts

Item

Function

Copy

Copies the chart to a clipboard.

Save Image As...

Saves the chart as an image file. Set the resolution and dimensions of
the image and then select the file type (PNG, GIF, JPG, TIF, or BMP).

Page Setup...

Selects a page setup for printing.

Print...

Prints the chart.

Set Scale to Default

Shows all of the data in the bar chart. Scroll bars display if there are too
many data points/samples to display in the chart frame.

Chart Settings

Opens the Chart Settings dialog box in which you can modify thechart’s display options including:n Chart and axis titles
n Chart and axis font and sizen Axis scalen Legend position

Chart tools also appear in each chart in the Data Analysis window. All charts display these tools:
Copy to Clipboard — copies the contents of the chart view to the clipboard.
Chart Settings — opens the Chart Settings dialog box in which you can modify the chart’s display
options.
Export — opens the Export Options dialog box, from which you can modify the resolution and size of thegraph and save it to a specified location as one of the following file types:
n

.bmp

n

.jpg

n

.png

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Charts

Bar Chart Tools
In addition to the chart tools, bar charts display the following tools:
Sort — sorts the targets and samples alphabetically or in reverse alpha order.
Color Settings — opens the Color Settings dialog box, in which you can change the color of the targets
and samples.
For more information about these tools, see Changing and Annotating the Chart View on page 244.

Amplification Chart Tools
In addition to those listed above, amplification charts display the following tools:
Trace Styles — opens the Trace Styles dialog box, in which you can modify the appearance of traces in
the amplification chart.
Baseline Threshold — opens the Baseline Threshold dialog box, in which you can modify the default
baseline for selected wells or change the threshold for each fluorescence curve in the amplification chart.

Copying Chart Data to the Clipboard
You can copy the contents of the chart view and paste it into any application that accepts bitmap image
files.

To copy chart data to the clipboard
1.

From the chart tools, select the Copy to Clipboard icon.

2.

Open an application that accepts bitmap images, for example Microsoft Word.

3.

Right-click and select Paste to paste the bitmap image from the clipboard into the application.

Changing the Chart Display Settings
Use the Chart Settings dialog box to change the titles, fonts and sizes, axis scale, and legend location forthe displayed chart. Changes that you make apply to the displayed chart only and are saved with thechart.

To change chart display settings
1.

From the chart tools, click Chart Settings.
The Chart Settings dialog box appears.

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Chapter 10 Data Analysis Overview

2.

3.

Select the Chart And Axes tab to:nType a title for the chart.

nType a new title for the x-axis and angle the labels.

nType a new title for the y-axis.

Select the Fonts tab to change the chart’s font and font size.
Tip: By default, the font size autoscales as the chart size changes. Select Change Font Size to
set a static font size for each label type.

4.

5.

Select the Axes Scale tab to:n

Clear x- and y-axis autoscaling and specify minimum and maximum scaling values.

nChoose to display grid lines or tick marks on the graph.

Select the Legend tab to:nChoose to hide the chart legend.

nChange the default position of the chart legend.

Note: When the legend is positioned to the left or right of the chart, it displays only the first ten
fluorophores in the chart.
6.

Click Apply at any time to view chart setting changes without saving the changes.

7.

Click OK to save the changes and return to the chart.

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Exporting the Chart
Use this dialog box to modify the width, height, and resolution of the graph to export it in one of thefollowing file formats:
n

.bmp

n

.jpg

n

.png

You can then use the exported graph to display your results in poster sessions, Microsoft PowerPoint
presentations, and professional journals.
Note: Consider the following when modifying the settings:nnMaximum and minimum width and height limitsoAt 72 dpi: 0.1–83 ino

At 96 dpi: 0.1–62 ino

At 150 dpi: 0.1–40 ino

At 300 dpi: 0.1–20 ino

At 600 dpi: 0.1–10 ino

At all resolutions: 2–6,000 pixels

Aspect ratio is based on width.

To export the chart
1.

From the chart tools, click Export.
The Export Preview dialog box appears.

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Chapter 10 Data Analysis Overview

2.

Modify the settings for the display as required.

3.

Click Export.

4.

In the Export dialog box, do the following:

5.

a.

(Optional) Navigate to a folder in which to save the chart file.

b.

Type a name for the file and choose a file type from the dropdown list.

Click Save to save the chart file.

Modifying the Baseline Threshold Settings
In Single Threshold mode, you can adjust the threshold for a fluorophore by clicking on the threshold linein the Amplification chart and moving the mouse pointer vertically. Alternatively, you can specify an exact
crossing threshold for the selected fluorophore.
Tip: You can specify a cycle range to determine the baseline for all data files in the Data Analysis tab
in User > User Preferences.

To adjust the begin and end baseline cycle for each well1.

In the Quantification tab, select a single fluorophore under the Amplification chart.

2.

From the chart tools, select Baseline Threshold.
The Baseline Threshold dialog box appears.

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Charts

3.

4.

In the Baseline Cycles section, do one of the following:n

To select one well, click its row number.

n

To select multiple adjacent wells, click the row number of the first well and drag down the column
to the final well.

n

To select multiple nonadjacent wells, press the Control key and click the row number of eachtarget well.

n

To select all wells, click the top left corner on the table.

Adjust the Baseline Begin cycle and Baseline End cycle for all selected wells, or change the Beginand End cycle number at the bottom of the spreadsheet.
Tip: To revert the settings back to the last saved values, click Reset All User Defined Values.

5.

Click OK to save the changes and return to the chart.

To specify a cycle range for all data filesuIn the Home or Plate Editor window, select User > User Preferences and choose the Data Analysis
tab.

Sorting Target, Sample, and Biological Group Data
Note: This option is available on gene expression charts only.
By default, the Targets, Samples, and Biological Groups lists appear in alphabetical order. Use the Sort
dialog box to sort the display in reverse alpha order or to manually move a term to a different position in
the list.

To sort target, sample, and biological group data
1.

From the chart tools, click Sort.
The Gene Expression Chart Sorting dialog box appears.

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Chapter 10 Data Analysis Overview

2.

In the dialog box, click Z-A to sort the list in reverse alphabetical order.

3.

To manually move a term, select it and click the appropriate button between the charts:

4.

nClick the Up or Down arrow to move the selected term one position.

n

Click the Up or Down bar arrow to move the selected term to the top or bottom of the list.

Click OK to save the changes and return to the Gene Expression tab.

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Charts

Changing the Target and Sample Color Settings
Note: This option is available on gene expression charts only.
Use the Color Settings dialog box to change the color of a target or sample, or to remove the item from thegraph.

To change color settings
1.

From the chart tools, select Color Settings.
The Color Settings dialog box appears.

2.

To change the display color for a target or sample, click its color in the Color column.

3.

In the Color dialog box that appears, select a new color and click OK.

4.

To remove the item from the gene expression graph, clear its checkbox in the Show Chart column.
Tip: To clear all items from the gene expression graph, clear the Show Chart checkbox in thecolumn head.

5.

(Optional) By default, the bar chart color appears in gradient form. To display the color in solid form,
select Use Solid Colors.

6.

Click OK to save the changes and return to the Gene Expression tab.

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Chapter 10 Data Analysis Overview

Magnifying an Area in the Chart
To magnify an area of the chartu

Click and drag across the chart and then click Zoom. The software resizes the chart and centers it on
the selected area.
Note: The Bar Chart does not require you to click the Zoom pop-up command.

To reset the chart to full viewu

Right-click in the chart and select Set Scale to Default.

Copying Charts into a Microsoft File
You can copy data charts into Microsoft Word, Excel, or PowerPoint documents. The image resolution
corresponds to that of the screen from which the image was obtained.

To copy charts into a Microsoft file1.

In the Data Analysis window, click Copy To Clipboard in the upper right corner of the chart’s pane.

2.

Open a blank Microsoft file and paste the contents from the clipboard.

Common Right-Click Menu Items for Charts
Table 15 lists the right-click menu items that are available on charts. Some of the items are present for all
charts, including items to change how the data are displayed or to easily export data from a chart.
Table 15. Right-click menu items for charts

Item

Function

Copy

Copies the chart into the clipboard.

Save Image As

Saves the image at a specified size, resolution, and file type including PNG(default), JPG, and BMP.

Page Setup

Displays print setup options.

Print

Prints the chart.

Set Scale to Default

Returns the chart to its default view after magnifying the chart.

Chart Options

Opens the Chart Options window to change the chart, including its title, selecting
limits for the x and y axes, and showing grid lines and minor ticks in the axes.

Note: Menu items that apply to specific charts are described in Chapter 11, Data Analysis Details.

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Spreadsheets

Spreadsheets
The spreadsheets shown in Data Analysis include options for sorting and transferring the data. Sort thecolumns by one of these methods:nClick and drag a column to a new location in the selected table.

n

Click the column header to sort the data in ascending or descending order.

To sort up to three columns of data in the Sort window
1.

Right-click in the spreadsheet and select Sort.

2.

In the Sort dialog box, select the first column title to sort. Sort the data in ascending or descendingorder.

3.

Select a second or third column to sort and choose Ascending or Descending.

4.

Click OK to sort the data or click Cancel to stop sorting.

Tip: Highlight the data on the associated charts and well selector by holding the mouse pointer over acell. Click in a cell to copy and paste its contents into another software program.

Common Right-Click Menu Items for Spreadsheets
Table 16 lists the right-click menu items available on any spreadsheet view.
Table 16. Right-click menu items for spreadsheets

Item

Function

Copy

Copies the contents of the selected wells to a clipboard, then paste
the contents into a spreadsheet such as Excel.

Copy as Image

Copies the spreadsheet view as an image file and paste it into a filethat accepts an image file, such as text, image, or spreadsheet files.

Print

Prints the current view.

Print Selection

Prints the current selection.

Export to Excel

Exports the data to an Excel spreadsheet.

Export to Text

Exports the data to a text editor.

Export to CSVExports the data to a .csv file.

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Chapter 10 Data Analysis Overview

Table 16. Right-click menu items for spreadsheets, continued

Item

Function

Export to Xml

Exports the data to an .xml file.

Export to Html

Exports the data to an .html file.

Find

Searches for text.

Sort

Sorts the data in up to three columns.

Select Columns

Selects the columns that will be displayed in the spreadsheet.

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ExportExport
CFX Maestro Dx SE provides several export options from the Export dropdown menu:nExport All Data Sheetsn

Export RDML Filesn

Custom ExportnExport to LIMS FoldernManual Export

Exporting All Data Sheets
You can export all the spreadsheet views from every tab of CFX Maestro Dx SE into individual files.

To export all data sheetsu

Select Export > Export All Data Sheets and then select the file type you want:n

CSV (*.csv)

nText (*.txt)

nExcel Workbook ( *.xlsx)
Exported analyses are saved in multiple Excel Workbook files with one analysis data worksheet
tab per file. When an analysis includes multiple fluorophores, the data from each fluorophore isexported to a separate worksheet tab.

n

Excel Workbook - combined ( *.xlsx)
Exported analyses are saved to single Excel Workbook file that includes multiple worksheettabs, one for each analysis data set.

n

Excel 97 - 2003 ( *.xls)
Important: Your computer must have Microsoft Excel installed in order for you to export
data to a Microsoft Excel spreadsheet.

nXml (*.xml)

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Chapter 10 Data Analysis Overview

Exporting RDML Files
RDML is a structured and universal data standard for exchanging quantitative PCR (qPCR) data. The
data standard is a text file in Extensible Markup Language (.xml) format. Refer to the International RDML
Consortium website (www.rdml.org) for additional information about the RDML data exchange format.
Important: Exported RDML files include analysis data with the baseline settings that you apply in the
Data Analysis window. For more information regarding baseline settings, see Baseline Settings on
page 182.
Note: Save the RDML file as version 1.1 if you are using version 2.3 or higher of qbase+ software.

To export an RDML file1.

Select Export > Export RDML Files and select RDML v1.1 or RDML v1.0 from the list that appears.
The Save As dialog box appears.

2.

In the Save As dialog box, specify a file name and location in which to save the RDML file.

3.

Click OK to save the export file.

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ExportCreating a Custom Export FileTo create a custom export file1.

Select Export > Custom Export. The Custom Export dialog box appears.

2.

Select the export format from the dropdown list that appears.

3.

Select the checkboxes for the items to export.

4.

(Optional) Click Customize Column Names to change column names.

5.

Click Export. The Save As dialog box appears.

6.

In the Save As dialog box, specify a file name and location in which to save the exported file.

7.

Click OK to save the export file.

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Chapter 10 Data Analysis Overview

Exporting to a LIMS Folder
You can export data into a LIMS-compatible file format. For more information about creating, managing,and using LIMS files, see Appendix C, LIMS Integration.

To export data in LIMS format
1.

Select Export > Export to LIMS Folder.
The Save As dialog box appears.

2.

In the Save As dialog box, specify a file name and location in which to save the exported file.

3.

Click OK to save the export file.

Exporting Seegene-Formatted Data
You can export the data from all spreadsheet views to Excel files structured specifically for use bySeegene, Inc.
Tip: You can also automatically start the Seegene Viewer when the export completes. See Tools
Menu Commands on page 63 for more information.

To export data in a Seegene-specific format
1.

Select Export > Manual Export.
The Browse For Folder dialog box appears.

2.

In the Browse For Folder dialog box, specify a folder location in which to save the exported Seegeneformatted Excel (.xlsx) files.
The analyses are exported in multiple Excel Workbook files with one analysis data worksheet tab per
file.

3.

Click OK to save the export files.

202 | CFX Maestro Dx SE SoftwareChapter 11 Data Analysis Details
The CFX Maestro Dx Software, Security Edition Data Analysis window comprises multiple tabs fromwhich to view data. This chapter explains these tabs in detail.
Tip: You can choose which tabs to view in the Data Analysis window using the View menu. The
customized layout is saved with the data file.

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Chapter 11 Data Analysis Details

Quantification Tab
Use the data in the Quantification tab to set the data analysis conditions, including the baseline settings
for individual wells and the threshold settings. The Quantification tab displays data in these four views:nAmplification chart — displays the relative fluorescence units (RFU) for each well at every cycle.
Each trace in the chart represents data from a single fluorophore in one well.

nStandard curve — appears only if the run includes wells designated as sample type standard (Std).
The standard curve displays the threshold cycle plotted against the log of the starting quantity. The
legend displays the Reaction Efficiency (E) for each fluorophore in the wells with a Standard sample
type.

n

Well selector — selects the wells with the fluorescence data you want to show.

n

Spreadsheet — displays a spreadsheet of the data collected in the selected wells.

Fluorophore Options
To display fluorophore data in the Quantification tab charts and spreadsheets, select the target
fluorophore(s) below the Amplification chart. To hide the fluorophore data in the data analysis window,
clear its checkbox.

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Quantification Tab

Trace Styles Dialog Box
Using the Trace Styles dialog box, you can adjust the appearance of traces in the amplification and melt
curve charts in the Quantification and Melt Curve tabs. You can then preview the changes in the wellselector that appears in the Trace Styles dialog box.

To adjust trace styles
1.

Select only one fluorophore under the Amplification chart.

2.

To open the Trace Styles dialog box, do one of the following:n

Click Trace Styles in the Amplification chart.

nSelect Settings > Trace Styles in the Data Analysis menu bar.

nRight-click on a trace and select Trace Styles.

The Trace Styles dialog appears.

3.

In the Trace Styles dialog box, select a specific set of wells in the well selector in the bottom pane.
Alternatively, select wells that contain one sample type in the dropdown menu in the Wells column.

4.

Do any of the following:n

To choose a color for the selected wells, click the box in the Color column.

n

To assign a symbol to the selected wells, select a symbol from the Symbol dropdown list.

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Chapter 11 Data Analysis DetailsnnTo quickly color the wells by button label, click the appropriate quick set:oRandom by WelloRandom by Replicateo

Use Fluor Colorso

Use Target Colorso

Use Sample Colors

To assign well labels, choose either Sample Type, Target Name, Sample Name, or Symbol.

Log Scale Option
Select Log Scale below the Amplification chart to view the fluorescence traces in a semilog scale:

Tip: To magnify any area of the chart, drag across the target area. To return to a full view, right-clickon the chart and select Set Scale to Default.

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Quantification Tab

Standard Curve Chart
The software creates a Standard Curve chart in the Quantification tab if the data include sample types
defined as Std for at least one fluorophore in the run.

The Standard Curve chart displays the following information:nName for each curve (the fluorophore or target).

nColor of each fluorophore or target.

n

Reaction efficiency (E). Use this statistic to optimize a multiplex reaction and to equalize the data fora standard curve.
Note: The reaction efficiency describes how much of your target is being produced with eachcycle in the protocol. An efficiency of 100% indicates that you are doubling your target with eachcycle.

n

Coefficient of determination, R2 (written as R^2). Use this statistic to determine how correctly the line
describes the data (goodness of fit).

n

Slope

n

y-intercept

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Chapter 11 Data Analysis Details

Amplification Chart Menu Options
In addition to the common right-click menu options for charts (see Common Right-Click Menu Items for
Charts on page 196), Table 17 lists the menu options available only on the Amplification chart.
Table 17. Amplification chart right- and left-click menu items

Menu Option

Function

Well XX, Fluor Target

Displays only this well, removes this well from view, sets color for thistrace, or excludes this well from analysis.

Selected Traces

Displays only these wells, removes these wells from view, sets color forthese traces, or excludes these wells from analysis.

Show Threshold Values

Displays the threshold value for each amplification curve on the chart.

Trace Styles

Opens the Trace Styles window to change trace styles that appear on the
Quantification and Melt Curve tabs.

Baseline Thresholds

Opens the Baseline Thresholds window to change the baseline or
thresholds of each fluorophore (changes appear in the Amplification chartin the Quantification tab).

Quantification Tab Spreadsheet
Table 18 defines the data displayed in the spreadsheet in the Quantification tab.
Table 18. Quantification tab spreadsheet content

Information

Description

Well

Well position in the plate

Fluor

Fluorophore detected

TargetTarget Name loaded in the Plate Editor wells

Content

A combination of the Sample Type (required) and Replicate #(optional) loaded in the Plate Editor

SampleSample Name loaded in the Plate Editor wells

Cq

Quantification cycle for each trace

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Quantification Tab

Changing Target, Content, or Sample Data
You can change the data in the Target, Content, and Sample columns by editing the plate file using the
Plate Editor even after you run the experiment.

To change the data in the Content, Target, and Sample columnsuClick Plate Setup and select View/Edit Plate to open the Plate Editor.

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Chapter 11 Data Analysis Details

Quantification Data Tab
The Quantification Data tab displays the quantification data collected in each well. CFX Maestro Dx SE
displays the data in four different spreadsheet views:nResults — displays a spreadsheet of the data. This is the default view.

nStandard Curve Results — displays a spreadsheet of the standard curve data.

n

Plate — displays the data in each well as a plate map.

n

RFU — displays the RFU quantities in each well for each cycle.

Select each spreadsheet from the dropdown list that appears below the Quantification Data tab.

Results Spreadsheet
The Results spreadsheet displays data for each well in the plate.

Note: All Std. Dev (standard deviation) calculations apply to the replicate groups assigned in thewells in the Plate Editor window. The calculations average the Cq value for each well in the replicate
group.
Table 19 defines the data that appear in the Results spreadsheet.
Table 19. Results spreadsheet content

Information

Description

Well

Well position in the plate

Fluor

Fluorophore detected

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Quantification Data Tab

Table 19. Results spreadsheet content, continued

Information

Description

TargetAmplification target name (gene)Content

Sample type and Replicate #SampleSample description

Biological Set Name

Name of the biological set

Cq

Quantification cycle

Cq MeanMean of the quantification cycle for the replicate group

Cq Std. Dev

Standard deviation of the quantification cycle for the replicate group

Starting Quantity (SQ)

Estimate of the starting quantity of the target

Log Starting Quantity

Log of the starting quantity

SQ MeanMean of the starting quantity

SQ Std. Dev

Standard deviation of the starting quantity across replicates

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Chapter 11 Data Analysis Details

Standard Curve Results Spreadsheet
The Standard Curve Results spreadsheet displays the calculated standard curve parameters.

Table 20 defines the data that appear in the Standard Curve Results spreadsheet.
Table 20. Standard Curve Results spreadsheet contents

Information

Description

Fluor (or Target)

Fluorophore (or Target) detected

Efficiency %Reaction efficiency

Slope

Slope of the standard curve

Y-intercept

Point at which the curve intercepts the y-axis

R^2

Coefficient of determination

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Quantification Data Tab

Plate Spreadsheet
The Plate spreadsheet displays a plate map of the data for one fluorophore at a time.

To view data for a specific fluorophoreu

Click its tab at the bottom of the spreadsheet.

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Chapter 11 Data Analysis Details

RFU Spreadsheet
The RFU spreadsheet displays the relative fluorescence units (RFU) readings for each well acquired ateach cycle of the run. The well number appears at the top of each column and the cycle number appears
to the left of each row.

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Melt Curve Tab

Melt Curve Tab
For DNA-binding dyes and noncleavable hybridization probes, the fluorescence is brightest when the two
strands of DNA anneal. Therefore, as the temperature rises towards the melting temperature (Tm),fluorescence decreases at a constant rate (constant slope). At the Tm there is a dramatic reduction in the
fluorescence with a noticeable change in slope. The rate of this change is determined by plotting thenegative first regression of fluorescence versus temperature (-d(RFU)/dT). The greatest rate of change influorescence results in visible peaks and represents the Tm of the double-stranded DNA complexes.
CFX Maestro Dx SE plots the RFU data collected during a melt curve as a function of temperature. To
analyze melt peak data, the software assigns a beginning and ending temperature to each peak bymoving the threshold bar. The floor of the peak area is specified by the position of the melt threshold bar.
A valid peak must have a minimum height relative to the distance between the threshold bar and theheight of the highest peak.
The Melt Curve tab displays the Tm (melt temperature) of amplified PCR products in four views:nMelt Curve — displays the real-time data for each fluorophore as RFUs per temperature for each
well.

n

Melt Peak — displays the negative regression of the RFU data per temperature for each well.

n

Well selector — displays wells to show or hide the data.

nPeak spreadsheet — displays the data collected in the selected well.
Note: This spreadsheet displays up to two peaks for each trace. To see more peaks, click theMelt Curve Data tab.

Table 21 defines the data that appear in the Melt Curve spreadsheet.

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Chapter 11 Data Analysis Details

Table 21. Melt Curve spreadsheet contents

Information

Description

Well

Well position in the plate

Fluor

Fluorophore detected

Content

A combination of Sample type and Replicate number

SampleName of sample loaded in the Plate Editor

Melt Temp

The temperature of the melt peak for each well
Note: Only the two highest peaks appear in this spreadsheet.

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Melt Curve Tab

Adjusting Melt Curve Data
To adjust the Melt Curve datau

Do any of the following:n

Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks in data
analysis.

nSelect Positive in the Peaks dropdown menu to show the spreadsheet data for the peaks above
the Melt Threshold line or select Negative to view the spreadsheet data for the peaks below theMelt Threshold line.

nOpen the Trace Styles window to change the color of the traces in the Melt Curve and Melt Peakcharts.

n

Select a number in the Step Number selector to view the Melt Curve data at another step in the
protocol. The list shows more than one step if the protocol includes plate reads in more than one
melt curve step.

n

Select wells in the well selector to focus on subsets of the data.

n

Select a well group to view and analyze a subset of the wells in the plate. Select each well groupby name in the Well Group dropdown menu in the toolbar.

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Chapter 11 Data Analysis Details

Melt Curve Data Tab
The Melt Curve Data tab displays the data from the Melt Curve tab in multiple spreadsheets that include
all the melt peaks for each trace. CFX Maestro Dx SE offers four spreadsheet options in which to view themelt curve data:nMelt Peaks — displays all the data, including all the melt peaks, for each trace. This is the default
view.

n

Plate — displays a view of the data and contents of each well in the plate.

n

RFU — displays the RFU quantities at each temperature for each well.

n-d(RFU)/dT — displays the negative rate of change in RFU as the temperature (T) changes. This is a
first regression plot for each well in the plate.

Select each spreadsheet from the dropdown list that appears below the Melt Curve Data tab.

Melt Peaks Spreadsheet
The Melt Peaks spreadsheet displays all melt curve data.

Table 22 on page 219 defines the data that appear in the Melt Peaks spreadsheet.

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Melt Curve Data Tab

Table 22. Melt Peaks spreadsheet content

Information

Description

Well

Well position in the plate

Fluor

Fluorophore detected

Content

Sample Type listed in the Plate Editor window

TargetAmplification target (gene)

SampleSample Name listed in the Plate Editor window

Melt Temperature

Melting temperature of each product, listed as one peak (highest) per
row in the spreadsheet

Peak Height

Height of the peak

Begin Temperature

Temperature at the beginning of the peak

End Temperature

Temperature at the end of the peak

Plate Spreadsheet
The Plate spreadsheet displays melt curve data in a plate format.

Note: To adjust the peak that the software calls, adjust the threshold line in the Melt Peak chart on
the Melt Curve tab.
Table 23 on page 220 defines the data that appear in the Plate spreadsheet.

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Chapter 11 Data Analysis Details

Table 23. Plate spreadsheet content

Information

Description

Content

Combination of Sample Type (required) and Replicate # (optional)

SampleSample description

Peak 1First melt peak (highest)

Peak 2

Second melt peak (lower)

RFU Spreadsheet
The RFU spreadsheet displays the fluorescence for each well at each cycle acquired during the melt
curve.

Table 24 defines the data displayed in the RFU spreadsheet.
Table 24. RFU spreadsheet content

Information

Description

Well number (A1, A2, A3, A4, A5)

Well position in the plate for the loaded wells

Temperature

Melting temperature of the amplified target,
plotted as one well per row and multiple wells
for multiple products in the same well

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Melt Curve Data Tab-d(RFU)/dT Spreadsheet
The -d(RFU)/dT spreadsheet displays the negative rate of change in RFU as the temperature (T)changes.

Table 25 defines the data that appear in the -d(RFU)/dT spreadsheet.
Table 25. -d(RFU)/dT spreadsheet content

Information

Description

Well number (A1, A2, A3, A4, A5)

Well position in the plate for the loaded wells

Temperature -d(RFU)/dT

Negative rate of change in RFU as temperature (T) changes

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Chapter 11 Data Analysis Details

End Point Tab
Open the End Point tab to analyze final relative fluorescence units (RFUs) for the sample wells. The
software compares the RFU levels for wells with unknown samples to the RFU levels for wells withnegative controls and “calls” the unknown positive or negative. Positive samples have an RFU value thatis greater than the average RFU value of the negative controls plus the cut off value.

To analyze the end-point data, the plate must contain negative controls or the software cannot make thecall.
nRun a Quantification protocol — set up a standard protocol. After completion of the run, open the
Data Analysis window, adjust the data analysis settings in the Quantification tab, and then click the
End Point tab to pick an end point cycle.

n

Run an End Point Only protocol — load the End Point Only protocol in the Plate tab of the Run Setup
window, select or create a plate, and start the run

The End Point tab displays the average RFU values to determine whether the target was amplified by thelast (end) cycle. Use these data to determine whether a specific target sequence is present (positive) in asample. Positive targets have higher RFU values than the cutoff level you define.

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End Point Tab

Tip: To create an end point protocol, open the Protocol tab (Run Setup window) and selectRun > End Point Only Run.
When the run completes, the data file opens to the End Point tab, which comprises the following sections:nSettings — adjusts data analysis settings.

nResults — displays the results immediately after you adjust the settings.

nWell Selector — selects the wells with the end-point data you want to show.

n

RFU spreadsheet — displays the end RFU collected in the selected wells.

Results Data
The Results section displays the following data:nLowest RFU value — lowest RFU value in the datanHighest RFU value — highest RFU value in the datan

Negative Control Average — average RFU for the wells that contain negative controlsn

Cut Off Value — calculated by adding the tolerance (RFU or Percentage of Range listed in the
Settings) and the average of the negative controls. Samples with RFUs that are greater than the
cutoff value will be called “Positive.” To adjust the cutoff value, change the RFU or Percentage ofRange

The Cut Off Value is calculated using this formula:
Cut Off Value = Negative Control Average + Tolerance
Select a tolerance by one of these methods:n

RFUs (default) — select this method to use an absolute RFU value for the tolerance. The minimum
RFU tolerance value is 2. The maximum is the absolute value of the highest RFU value minus theabsolute value of the lowest RFU value. The default RFU tolerance value is 10% of the total RFU
range.

n

Percent of Range — select this method to use a percentage of the RFU range for the tolerance. The
minimum percent of range is 1%. The maximum percent of range is 99%. The default percent ofrange is 10%.

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Chapter 11 Data Analysis Details

Adjusting the End Point Data Analysis
To adjust the data in the End Point tabu

Do any of the following:nChoose a fluorophore from the dropdown list.

nChoose an End Cycle to Average value to set the number of cycles with which to calculate theaverage end-point RFU.

n

Select RFUs to view the data in relative fluorescence units.

nSelect Percentage of Range to view the data as a percentage of the RFU range.

nSelect wells in the well selector to focus on subsets of the data.

n

Select a well group to view and analyze a subset of the wells in the plate. Select each well groupby name in the Well Group dropdown menu in the toolbar.

RFU Spreadsheet for End Point Analysis
Table 26 defines the data that appear in the RFU spreadsheet in the End Point tab.
Table 26. RFU End Point spreadsheet contents

Information

Description

Well

Well position in the plate

Fluor

Fluorophore detected

Content

Combination of the Sample type and Replicate #End RFU

RFU at the end-point cycle

Call

Positive or Negative, where positive samples have an RFU value greaterthan the average RFU of the negative controls plus the Cut Off Value

SampleSample Name loaded in the Plate Editor

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Allelic Discrimination Tab

Allelic Discrimination Tab
The Allelic Discrimination tab assigns genotypes to wells with unknown samples. Use these data toidentify samples with different genotypes, including Allele 1, Allele 2, Heterozygote, No Call (noamplification), or Undetermined.
Note: The data for allelic discrimination must come from multiplex runs with at least two fluorophores.
Each fluorophore identifies one allele in all samples.

Allelic discrimination analysis requires the following minimal well contents:n

Two fluorophores in each welln

NTC (no template control) samples for optimized data analysis

CFX Maestro Dx SE offers four options in which to view allelic discrimination data:nAllelic Discrimination chart — displays the data in a graph of RFU for Allele 1/Allele 2. Each point in
the graph represents data from both fluorophores in one well. You can toggle between Cartesian andPolar coordinates by selecting and clearing the Polar Coordinates checkbox. Cartesian Coordinates
represents RFU for Allele 1 on the x-axis and RFU for Allele 2 on the y-axis. Polar Coordinates
represents the angle on the x-axis and distance between the origin and the RFU on the y-axis(median of all NTC).

n

Well spreadsheet — displays the allelic discrimination data collected in each well of the plate.

User Guide | 225

Chapter 11 Data Analysis DetailsnWell selector — selects the wells with the allelic data you want to show.

n

Selected Fluorophores panel — changes the x- and y-axis labels in the Allelic Discrimination chart,
the cycle to analyze, and whether to display the call map.

Adjusting Data for Allelic Discrimination
The software automatically assigns a genotype to wells with unknown samples based on the positions of
the NTCs and the angle and distance of the unknown data points from the NTCs.

To adjust allelic discrimination datau

Do any of the following:n

To display polar coordinates, select the checkbox in the Allelic Discrimination chart.

nTo view another fluorophore, choose it from the dropdown list in the Selected Fluorophores
panel.

n

To change a call, drag across the data point(s) in the Allelic Discrimination chart and choose an
option in the Selected Wells list:oAllele 1oAllele 2oHeterozygoteoUndeterminedo

No CalloAuto Call

Tip: Select Auto Call to revert to the default call.

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Allelic Discrimination Tab

Chart Menu Options
In addition to the common right-click menu options for charts (see Common Right-Click Menu Items for
Charts on page 196), Table 27 lists the menu options available on the Allelic Discrimination chart.
Table 27. Allelic Discrimination chart right- and left-menu options

Menu Option

Function

Zoom

Focuses the chart view to the area selected (by clicking anddragging the cursor in the chart).
Tip: To restore the zoom to show all data points, right click andselect Set Scale to Default.

Well

For the selected well, options are: display only this well, remove thiswell from view, set color for this trace, or exclude this well fromanalysis.

Selected Wells

For the selected wells (selected by clicking and dragging the cursor
in the chart), options are: display only these wells, remove thesewells from view, set color for these traces, or exclude these wellsfrom analysis.

Allelic Discrimination Spreadsheet
Table 28 defines the data that appear in the Allelic Discrimination spreadsheet.
Table 28. Allelic Discrimination spreadsheet contents

Information

Description

Well

Well position in the plate

SampleSample name description

Call

Identity of the allele, including automatic Allele 1, Allele 2,
Heterozygote, No Call, or UndeterminedType

Auto (Automatic) or Manual, describes how the call was made.
Automatic indicates that the software selected the call. Manual
indicates that the user chose the call

RFU1

RFU for Allele1

RFU2

RFU for Allele2

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Chapter 11 Data Analysis Details

Custom Data View Tab
The Custom Data View tab simultaneously displays multiple panes in a customizable format.
The Load a Preset View dropdown list offers a selection of display format templates. The default view
displayed is dependent on the file being analyzed. For example, if Melt Curve data are present, theAmp+Melt default view appears.

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Custom Data View Tab

Creating a Custom Data View
To create a custom data viewu

Do any of the following:nSelect an alternate preset view from the dropdown list.

nSelect another chart view from the dropdown list located at the top of each individual pane.

nChange the number of rows and columns in the tab.

nChange individual pane dimensions. Drag the bars at the periphery of each pane.

Click Save as Preset to save the customized as a preset template. Click Manage Presets to delete,rename, or restore existing preset views.

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Chapter 11 Data Analysis Details

QC Tab
Use the QC tab to quickly assess the quality of the run data based on the rules defined in the QC tab in theUser Preferences window.
CFX Maestro Dx SE offers four options in which to view the QC data:n

Amplification chart — displays the RFU for each well at every cycle. Each trace in the chart
represents data from a single fluorophore in one well.

n

QC rules table — displays the available QC rules and the settings that define each rule. Applied QC
rules are indicated by a checkmark.

n

Well selector — selects the wells with the fluorescence data you want to show.

n

QC rule summary pane — displays the selected QC rule and highlights wells that fail the rule.

Changing QC Criteria
To change QC criteriau

Select or clear the Use checkbox for the rule to include or exclude from QC.

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QC TabExcluding Wells That Fail QC
CFX Maestro Dx SE displays wells that fail QC criteria in the Results column in the QC rules table and in
the summary pane.

To exclude wells that fail QC criteriau

Select Exclude Wells for each well to exclude.

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Chapter 11 Data Analysis Details

Run Information Tab
The Run Information tab displays the protocol and other information about each run. Use this tab to do thefollowing:nView the protocol.

nEnter or edit notes about the run.

nEnter or edit the ID or bar code for the run.

nView events that might have occurred during the run. Use these messages to help troubleshoot a run.

Tip: Right-click the Protocol to copy, export, or print it. Right-click the Notes, ID/Bar Code, or Other
panes to undo, cut, copy, paste, delete, or select the text.

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Data Analysis Reports

Data Analysis Reports
The Report dialog box displays information about the current data file in the Data Analysis window. To
open a report, select Tools > Reports or click Reports on the toolbar.
The Report dialog contains the following sections:nMenu and toolbar — provides options to format, save, and print the report or template.

n

Options list (top left side of the dialog box) — provides options to display in the report.

nOptions pane (bottom left side of the dialog box) — displays text boxes in which you can enter
information about a selected option.

nPreview pane (right side of the dialog box) — displays a preview of the current report.

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Chapter 11 Data Analysis Details

Data Analysis Report Categories
Table 29 lists all the options available for a data analysis report, depending on the type of data in the Data
Analysis window.
Table 29. Data analysis report categories in the options list

Category

OptionDescription

HeaderTitle, subtitle, and logo for the report
Report Information

Run date, user name, data file name,data file path, and selected well group

Audit Information

Supplementary information required forauditing, including signatures

Notes

Notes about the data report

Run Information

Run date, user name, data file name,

Run Setup

data file path, and selected well group
ProtocolText view of the protocol steps andoptions

Plate Display

Plate view of the information in each wellof the plate

Quantification
Analysis Settings

Data collection step number, analysis
mode, and baseline subtraction method

Amplification Chart

Amplification chart for runs that include
quantification data

Standard Curve Chart

Standard curve chart

Data

Spreadsheet listing the data in each well

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Data Analysis Reports

Table 29. Data analysis report categories in the options list, continued

Category

OptionDescription

Gene Expression — Bar Chart
Analysis Settings

Analysis mode, chart data, scaling option,and chart error

Chart

Copy of the bar chart

Target Names

Chart of target names

Sample Names

Chart of sample names

Data

Spreadsheet listing the data in each well

Target Stability

Chart of the target stability values

Box-and-Whisker Chart

Box-and-whisker chart

Dot Plot Chart

Dot plot chart

Gene Expression — Clustergram, and Scatter Plot
Analysis Settings

Settings for each chart type

Chart

Copy of the chart

Data

Spreadsheet listing the data in eachtargetGene Expression — ANOVA Data
ANOVA Settings

P-value threshold used in the analysis

ANOVA Results

Table of results from ANOVA and Tukey's
HSD post-hoc analysis

Melt Curve
Analysis Settings

Melt step number and threshold bar
setting

Melt Curve Chart

Melt curve chart

Melt Peak Chart

Melt peak chart

Data

Spreadsheet listing the data in each well

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Chapter 11 Data Analysis Details

Table 29. Data analysis report categories in the options list, continued

Category

OptionDescription

Analysis Settings

Fluorophores, cycle, and view call map

Allelic Discrimination Chart

Copy of the allelic discrimination chart

Data

Spreadsheet listing the data in each wellAnalysis Settings

Fluorophore, end cycles to average,

Allelic Discrimination

End Pointmode, lowest RFU value, highest RFU
value, and cut off value
Data

Spreadsheet listing the data in each well

Data

Spreadsheet listing the parameters for

QC Parameters

each QC rule236 | CFX Maestro Dx SE Software

Data Analysis Reports

Creating a Data Analysis Report
You can save the report layout as a template, which you can use again for similar reports.

To create a data analysis report1.

Make final adjustments to the well contents, selected wells, charts, and spreadsheets in the Data
Analysis window before creating the report.

2.

Select Tools > Reports in the Data Analysis menu bar to open the Report dialog box.

3.

Choose the options you want to include in the report. The report opens with default options selected.
Select or clear the checkboxes to change whole categories or individual options within a category.
Table 29 on page 234 lists the available options to display.
Note: The data that appear in the report depend on the current selections within the tabs of theData Analysis window. For example, a quantification run might not contain a standard curve, andtherefore those data do not appear in the Data Analysis window or in the data report.

4.

Change the order of categories and items in a report. Drag the options to the relative position. Items
can be reordered only within the categories to which they belong.

5.

(Optional) In the Report Options pane, enter information relevant to the selected option:nChoose a subset of information to display in the report.

nChoose specific settings for the selected option.

nChange the text to display for the selected option.

6.

Click Update Report to update the Report Preview with any changes.

7.

Print or save the report:

8.

a.

Click the Print Report button in the toolbar to print the current report.

b.

Select File > Save to save the report in PDF (Adobe Acrobat Reader file), MHT (Microsoft
document), or MHTML (Microsoft document) file format.

c.

Select a location in which to save the file.

d.

Select File > Save As to save the report with a new name or in a new location.

(Optional) Create a report template with the information you want. To save the current report settings
in a template, select Template > Save or Save As. Then load the report template the next time youwant to make a new report.

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Chapter 11 Data Analysis Details

Creating Well Group Reports
To create a well group report
1.

Select Tools > Well Group Reports in the Data Analysis window.

2.

In the Well Groups Reports dialog box, select the well groups, amplification steps, and melt steps toinclude in the report.

3.

Enter the path or navigate to the destination folder in which to save the report.

4.

(Optional) Select Choose a Report Template and navigate to the template file folder.

5.

(Optional) Select Open Destination Folder to open the folder and view the reports after they are
generated.

6.

Click Create Reports.

238 | CFX Maestro Dx SE SoftwareChapter 12 Gene Expression Analysis
With the use of stringently qualified controls in your reactions, you can use CFX Maestro Dx Software,
Security Edition to perform a gene expression run to normalize the relative differences in a target
concentration among samples. Typically, expression levels for one or more reference genes are used tonormalize the expression levels of a gene of interest. Reference genes take into account loading
differences or other variations represented in each sample and their expression levels should not beaffected in the biological system being studied.
Choose the Gene Expression tab in the Data Analysis window to evaluate relative differences between
PCR reactions in two or more wells. For example, you can evaluate relative numbers of viral genomes orrelative numbers of transfected sequences in a PCR reaction. The most common application for gene
expression study is the comparison of cDNA concentration in more than one reaction to estimate thelevels of steady state messenger RNA.
The software calculates the relative expression level of a target with one of these scenarios:nRelative expression level of a target sequence (Target 1) relative to another target (Target 2); forexample the amount of one gene relative to another gene under the same sample treatment.

nRelative expression level of one target sequence in one sample compared to the same target under
different sample treatment; for example, the relative amount of one gene relative to itself under
different temporal, geographical, or developmental conditions.

Plate Setup for Gene Expression Analysis
To perform gene expression analysis, the contents of the wells must include the following:n

Two or more targets — the two targets that represent different amplified genes or sequences in yoursamples.

n

One or more reference targets — at least one target must be a reference target for normalized
expression. Assign all reference targets in the Experiment Settings window to analyze the data in
Normalized Expression mode (ΔΔCq). Runs that do not contain a reference must be analyzed using
Relative Expression mode (ΔCq).

nCommon samples — your reactions must include common samples (minimum of two required) toview your data plotted in the Gene Expression tab. These samples should represent different

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Chapter 12 Gene Expression Analysis

treatments or conditions for each of your target sequences. Assign a control sample (optional) in theExperiment Settings window. If no control is selected, the software uses the lowest Cq as the control.
The requirements for Gene Expression setup in the Plate Editor depend on whether reaction contents are
singleplex PCR, with one fluorophore in the reactions, or multiplex PCR, with more than one fluorophorein the reactions.

Guided Plate Setup
If the plate setup of a data file does not contain the information required for analysis and the Gene
Expression tab is selected, the space normally occupied by the bar chart will contain instructions forentering this information. For normalized gene expression, complete the following steps:
1.

2.

Define Target and Sample names using any of the following:n

Plate Setup — opens the Plate Editor window.

n

Replace Plate File — opens the Select Plate browser, in which you can navigate to a previously
saved plate file with which to replace the current plate layout.

n

Replace PrimePCR File — opens the Select PrimePCR file dialog box, in which you cannavigate to a PrimePCR run file and apply it to the plate layout.

Select one or more reference targets and a control sample using the Experiment Settings dialog box.
If the plate layout already contains target and sample information, only the second step is required
and is highlighted orange. This step must be completed before normalized gene expression analysis
can occur.
Note: Data for the scatter plot and clustergram are displayed only if all of the requirements fornormalized gene expression listed under Plate Setup for Gene Expression Analysis are met.

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Gene Expression Charts

Gene Expression Charts
CFX Maestro Dx SE displays gene expression data in multiple views. Table 30 lists chart options
available in the software.
Table 30. Gene expression chart options

ButtonName

Function

Graphing

Displays normalized gene expression data in
one of the following views:
n Bar chart (the default)n Box and whisker chartn Dot plot chart

Clustergram

Displays the normalized expression data in ahierarchy based on the degree of similarity of
expression for different targets and samples.

Scatter Plot

Displays the normalized expression of targets
for a control versus an experimental sample.

ANOVA

Displays the results of one-way ANOVA on thegene expression data using the following R
packages to perform ANOVA and determine
Tukey results:
n Companion to Applied Regression (car)n Least-square means (lsmeans)

Reference Gene Selection Tool

(Available on the Study Analysis tab in the Gene
Study window) Identifies the tested reference
genes and categorizes them as Ideal,
Acceptable, or Unstable based on their stability.

PrimePCR Controls Analysis(Available on the Study Analysis tab in the Gene
Study window) Displays the results of the tested
samples.

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Chapter 12 Gene Expression Analysis

Graphing

The relative expression of targets is presented in these two views:
nGene Expression chart — displays the real-time PCR data as one of the following:o

ΔΔCq — relative normalized expression calculated using control samples and reference targets.

o

ΔCq — relative quantity of the target gene in a sample relative to a control sample.

See Changing and Annotating the Chart View on page 244 for more information about viewing data.
n

Spreadsheet — displays a spreadsheet of the gene expression data.
Tip: Right-click any chart or spreadsheet for options. Select View/Edit Plate from the Plate Setup
dropdown menu to open the Plate Editor and change well contents in the plate.
Tip: Select Sort from the right-click menu to rearrange the order of the Target and Sample
names in the chart.

Normalized Gene Expression
To normalize data, use the measured expression level of one or more reference genes as a normalization
factor. Reference genes are targets that are not regulated in the biological system being studied, such as
actin, GAPDH, or tubulin.

242 | CFX Maestro Dx SE SoftwareGraphing

To set up normalized gene expression (ΔΔCq) analysis
1.

Open a data file (.pcrd extension).

2.

Review the data in the Quantification tab of the Data Analysis window. Make adjustments to the data,such as changing the threshold and the analysis mode.

3.

Choose the Gene Expression tab.

4.

In the Gene Expression tab, click Experiment Settings.

5.

In the Experiment Settings dialog box, do the following:a.

Choose the Samples tab and select a control. When a control is assigned, CFX Maestro Dx SE
normalizes the relative quantities for all genes to the control quantity, which is set to 1.

b.

6.

Choose the Target tab and select reference genes. Gene expression analysis requires one
reference among the targets in your samples.

Select Normalized Expression (ΔΔCq) if it is not already selected, and then view the expression
levels in the Gene Expression tab.
Note: You can also use the Setup Wizard to set up the plate layout for normalized gene
expression analysis.

Relative Quantity
By definition, relative quantity (ΔCq) data are not normalized. This method is used to quantitate samples
that do not include any reference genes (targets). Typically, researchers are confident in one of thefollowing considerations when they set up their run:nEach sample contains the same amount of RNA or cDNA in each well.

n

Any variance in the amount of biological sample loaded will be normalized after the run by somemethod in the data analysis outside of the software. For example, a researcher might choose to divide
the relative quantity value by the normalizing factor, possibly the mass of nucleic acid loaded for eachsample, or the number of cells from which the nucleic acid was isolated.

To run a Relative Quantity (ΔCq) analysisu

In the Gene Expression tab, select Relative Quantity (ΔCq) from the Mode dropdown list in the rightpane.
Tip: To compare results to data from other gene expression runs, open a new gene study or add
a data file to an existing gene study.

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Chapter 12 Gene Expression Analysis

Changing and Annotating the Chart View
Using the charts toolbar menu commands and the data analysis chart tools, you can change the chartview, annotate each chart, and change the chart display. The charts toolbar appears between the chartand the data analysis spreadsheet at the bottom of the screen.

Chart Toolbar Tools
Tip: See Charts on page 188 for information about the chart tools that appear on the right side of data
analysis charts.
The toolbar below the charts provides quick access to annotation tools.

Table 31 lists the functions of the buttons in the charts toolbar.
Table 31. Charts toolbar

ButtonName

Function

Bar chart

Displays the relative expression of the targets.

Box and Whisker chart

Displays data as quartile ranges (see Box and Whisker
Chart Calculations on page 278 for calculation details).
Note: Available only if Analyze Using is set to BiologicalGroups Only.

Dot Plot chart

Displays the individual sample data points for each target.
Note: Available only if Analyze Using is set to BiologicalGroups Only.

Add Arrow

Draws an arrow on the active chart.

Add Circle

Draws a circle on the active chart

Add Text

Inserts a textbox on the active chart, in which you can add
text to identify items of interest in the chart.

Undo

Removes or reverts the last annotation performed on theactive chart.

244 | CFX Maestro Dx SE SoftwareGraphing

Table 31. Charts toolbar, continued

ButtonName

Function

Redo

Reverts the last Undo action performed on the active
chart.

Clear All

Clears all annotations on the active chart.

Sorting Target, Sample, and Biological Group Data
Note: This option is available on gene expression charts only.
By default, the Targets, Samples, and Biological Groups lists appear in alphabetical order. Use the Sort
dialog box to sort the display in reverse alpha order or to manually move a term to a different position in
the list.

To sort target, sample, and biological group data
1.

From the chart tools, click Sort.
The Gene Expression Chart Sorting dialog box appears.

2.

In the dialog box, click Z-A to sort the list in reverse alphabetical order.

3.

To manually move a term, select it and click the appropriate button between the charts:

4.

nClick the Up or Down arrow to move the selected term one position.

n

Click the Up or Down bar arrow to move the selected term to the top or bottom of the list.

Click OK to save the changes and return to the Gene Expression tab.

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Chapter 12 Gene Expression Analysis

Changing the Target, Sample, and Biological Group Color Settings
Use the Color Settings dialog box to change the color of a target, sample, or biological group, or remove
the item from the graph.

To change the target color settings
1.

In the right pane in the Gene Expression dialog box, verify that Sample appears in the X-Axis
dropdown list.

2.

In the Chart Tools, select Color Settings.
The Color Settings dialog box appears.

3.

To change the display color for a target, click its color in the Color column.

4.

In the Color dialog box that appears, select a new color and click OK.

5.

To remove a target from the gene expression graph, clear its checkbox in the Show Chart column.
Tip: To clear all targets, clear Show Chart in the column head.

6.

(Optional) By default, the bars appear in solid colors. To display the bars in gradient colors, clear Use
Solid Colors.

7.

Click OK to save the changes and return to the Gene Expression tab.

To change the sample or biological group color settings
1.

In the right pane on the Gene Expression dialog box, verify that Target appears in the X-Axis
dropdown list.

2.

Perform the steps in To change the target color settings on page 246.

Changing the Chart View
To change the current chart viewu

Select the toolbar menu command for the target view.

Note: The Gene Expression tab always opens displaying the data in default Bar Chart view.

246 | CFX Maestro Dx SE SoftwareGraphingExcluding Outlier Data Points
In the Dot Plot chart, you can easily view and exclude outliers from your analysis.

To exclude outlier data pointsu

In the Dot Plot chart, right-click the target outlier and select Exclude Well from Analysis.

The data point is removed from the Dot Plot chart and the well changes to gray in the Well Selector in the
Quantification tab.

To include an excluded outlier data pointu

In the Quantification tab, right-click the well in the Well Selector and select Well > Include in Analysis.

Viewing Data Point Details
To view data point detailsu

In the Box and Whisker plot or the Dot Plot, pause your cursor on an individual data point.

A tooltip displays, showing the sample name and its expression (relative quantity or normalized
expression, depending on the selected mode).

Annotating Charts
You can add arrows, circles, and text to each bar chart view to clearly communicate data. The annotations
are saved with the bar chart and appear in the exported and printed file. However, annotations made to
one chart view are not added to the other chart views.

To draw an arrow or circle on the chart
1.

In the bar chart toolbar, click the specific tool.

2.

Click in the bar chart and drag your cursor across the chart as necessary.

To add text to the chart
1.

In the bar chart toolbar, click Add Text.

2.

Click in the bar chart. A text box appears at that location.

3.

Add text in the text box.

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Chapter 12 Gene Expression Analysis

4.

Click anywhere on the chart to exit the text box.
Tip: Press Enter to add multiple lines to the text box.

To move an annotation
1.

Hover your cursor over the annotation. The icon changes to a pointing finger and the annotation
border is highlighted.

2.

Click the annotation and drag it to another position.

3.

Release the annotation to secure its position.

To undo an annotationu

Click Undo.
The most recently added annotation is removed.
Tip: You can undo the ten most recent annotations, one at a time.

To redo an annotationu

Click Redo.
The most recently removed annotation returns.
Tip: You can redo the ten most recent annotations, one at a time.

To delete an annotationuRight-click the annotation and select Delete.

248 | CFX Maestro Dx SE SoftwareGraphing

Adjusting Gene Expression Data
After selecting your analysis mode, normalized expression (∆∆Cq) or relative quantity (∆Cq), adjust thedata you view in the Gene Expression tab by changing the settings options to the right of the chart.
Tip: You set the default Gene Expression data options in the User Preferences dialog box (see Setting
Default Gene Expression Data File Parameters on page 85).

Graph Data
Set the y-axis value to Linear scale to enable graph data options. Graph data options allow you to present
the data in the graph with one of these options:nRelative to control — graph the data with the axis scaled from 0 to 1. If you assign a control in yourrun, select this option to quickly visualize upregulation and downregulation of the target.

nRelative to zero — graph the data with the origin at zero.

Analyze Using
Use the dropdown menu to select how data is analyzed and plotted. The options are:n

Samples Only — data are analyzed and plotted on a per sample basis.

nBiological Groups Only — data are analyzed and plotted for biological groups. The expression
displayed for the biological group is the geometric mean of the samples in that group.

n

Sample Biological Group — data are analyzed and plotted on a per sample basis with the biologicalgroup appended after the sample name. The P-values shown are calculated based on the biologicalgroup.

nBiological Group Sample — data are analyzed and plotted on a per sample basis with the biologicalgroup prepended before the sample name. The P-values shown are calculated based on the
biological group.

Control Sample
Use the dropdown menu to select a sample that will be used to normalize the Relative Quantity:

Annotate P-Values and P-Value Threshold
When Annotate P-Values is selected, the software displays an asterisk (*) on the bar chart above a target
if its P-value is below the selected threshold. The software automatically calculates the P-value bycomparing the sample’s expression level to the selected control sample’s expression level using astandard t-test. The P-value threshold range is 0.000—1.000.

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Chapter 12 Gene Expression Analysis

X-Axis Options
The x-axis option allows you to select the x-axis data of the Gene Expression chart:nTarget — graphs the target names on the x-axis.

nSample — graphs the sample names on the x-axis.

Y-Axis Options
The y-axis option allows you to show the Gene Expression chart in one of these three scales:nLinear — select this option to show a linear scale.
Tip: Setting the y-axis to Linear enables the Graph Data dropdown list, from which you canchoose to graph data relative to control or relative to zero.

n

Log 2 — select this option to evaluate samples across a large dynamic range.

n

Log 10 — select this option to evaluate samples across a very large dynamic range.

Scaling Options
Select Normalized Gene Expression (ΔΔCq) and set the to None to enable the scaling options in the Gene
Expression chart. Select one of these scaling options to calculate and present your data in a manner thatbest suits your run design:n

Unscaled — presents the unscaled normalized gene expression.

nHighest — scales the normalized gene expression for each target by dividing the expression level ofeach sample by the highest level of expression in all the samples.
This scaling option uses the scaled-to-highest formula.

nLowest — scales the normalized gene expression for each target by dividing the expression level ofeach sample by the lowest level of expression in all the samples.
This scaling option uses the scaled-to-lowest formula.

nAverage — scales the normalized gene expression for each target by dividing the expression level ofeach sample by the geometric mean of the expression levels for all the samples.
This scaling option uses the scaled-to-average formula.

Select an option for the type of error calculations (error bars) in the Gene Expression chart:

Chart Error Bar Multiplier
Select a multiplier for the error bars in the Gene Expression chart. Select one of these integers:n+/- 1 (default)

250 | CFX Maestro Dx SE SoftwareGraphing

n

2

n

3

The type of multiplier changes when you select the error bar:n

SEMs for standard error of the meann

Std Devs for standard deviations

Experiment Settings
Tip: This dialog box is also available in the Plate Editor. For more information, see Changing
Experiment Settings on page 140.
In the Experiment Settings dialog box, you can view or change the list of targets, samples, or biologicalgroups, select reference genes, select controls, or set the Gene Expression Analysis group to beanalyzed if biological groups have been added to the wells.

To open the Experiment Settings dialog box
u

In the Graphing tab, click Experiment Settings at the bottom of the right pane.
The Experiment Settings dialog box appears displaying the Targets tab.

To adjust Targets settingsu

In the Targets tab, do any of the following:n

To select a target as a reference for gene expression data analysis, select its name in the
Reference column.

n

To change the target’s color, click its cell in the Color column and change the color in the Colordialog box that appears.
The color change appears in the Gene Expression charts.

n

To use a previously determined efficiency value, clear the target’s checkbox in the Auto
Efficiency column and enter a number for the efficiency percentage of a target.
The software calculates the relative efficiency for a target using Auto Efficiency if the data for atarget include a standard curve.

To adjust the Sample settingsu

In the Samples tab, do any of the following:n

To select a sample as a control for gene expression data analysis, select its name in the Control
column.

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Chapter 12 Gene Expression Analysisn

To change the sample group’s color, click its cell in the Color column and change the color in the
Color dialog box that appears.
The color change appears in the Gene Expression charts.

n

To display the sample in the Gene Expression charts, select it in the Show Chart column.

n

To remove the sample from the Gene Expression charts, clear it in the Show Chart column.
Tip: The sample group’s data remain in the Results table.

To exclude a sample type from analysis calculationsuSelect its checkbox at the bottom of the Experiment Settings dialog box.
Note: This excludes controls and/or standards from gene expression analysis.

Right-Click Menu Options
Right-click on the gene expression chart to select the items shown in Table 32.
Table 32. Gene expression right-click menu items

Item

Function

Copy

Copies the chart to a clipboard.

Save Image As

Saves the chart as an image file. Set the resolution and dimensions of
the image and then select the file type (PNG, JPG, or BMP).

Page Setup

Selects a page setup for printing.

Print

Prints the chart.

Set Scale to Default

Show All displays all of the data in the bar chart. Scroll Bar displays ascroll bar if there are too many samples to display in the chart frame
while maintaining a minimum bar width.

Chart Settings

Opens the Chart Settings window to adjust the graph.

Sort

Sorts the order of samples or targets that appear on the chart x-axis.

Use Corrected Std Devs

Calculates the error bars using the corrected standard deviation
formula.

Use Solid Bar Colors

Displays solid bars in the chart.

X–Axis Labels

Displays x-axis labels horizontally or angled.

252 | CFX Maestro Dx SE SoftwareGraphing

Data Spreadsheet
Table 33 defines the data displayed in the Gene Expression Data Table.
Note: The values in the table are calculated based on the graph type and preferences selected in theright-hand pane.
Table 33. Description of information in the spreadsheet on the tab

Information

Description

TargetTarget name (amplified gene) selected in the Experiment Settings
window.

Biological Group

Sample and/or biological group name selected in the Experiment

Sample Biological Group

Settings window.

Biological Group Sample
Control

Control name selected in the Experiment Settings window. When
Analyze Using is set to Samples Only, Control is the sample selectedin the Experiment Settings window. When either Biological Groups
Only, Sample Biological Group, or Biological Group Sample isselected, control is the biological group selected in the ExperimentSettings window.

Relative Quantity or

Relative Quantity (ΔCq) or Normalized Gene Expression (ΔΔCq),

Expression

depending on the selected mode.

Relative Quantity or Expression

Standard error of the mean (SEM) or standard deviation (SD) of theSEM (or SD)

relative quantity or normalized expression, depending on the selected
option. Available only if Analyze Using is set to Samples Only, Sample
Biological Group, or Biological Group Sample.

Corrected Relative Quantity or

Corrected value calculation for SEM or SD of the relative quantity or

Expression SEM (or SD)

normalized expression, depending on the selected option. Available
only if Analyze Using is set to Samples Only, Sample Biological Group,or Biological Group Sample.

Mean Cq

Mean of the quantification cycle (not displayed if Analyze Using is set
to Biological Groups Only).

Cq SEM (or SD)

SEM or SD of the quantification cycle, depending on the selected
option (not displayed if Analyze Using is set to Biological Groups Only).

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Chapter 12 Gene Expression Analysis

Show Details Option
Table 34 defines the data displayed when Show Details is selected from the right-click menu of the bar
chart spreadsheet.
Table 34. Information in the bar chart spreadsheet with Show Details selected

Information

Description

Data Set

Fluorescence data from one fluorophore in the data file

Relative Quantity

Calculated relative quantity of samples

Relative Quantity SD

Standard deviation of the relative quantity calculation

Corrected Relative Quantity SD

Calculated standard deviation of the corrected relative quantity

Relative Quantity SEM

Standard error of the mean of the relative quantity calculation

Corrected Relative Quantity

Calculated standard error of the mean of the corrected relative quantity

SEM
Relative Quantity(lg)Log2 of the relative quantity that is used for statistical analysis

SD RQ(lg)Standard deviation of the relative quantity (log2)

SEM Expression(lg)Standard error of the mean of the expression (log2)

Unscaled Expression

Calculated unscaled expression

Unscaled Expression SD

Calculated standard deviation of the unscaled expression

Corrected Unscaled Expression

Calculated standard deviation of the corrected unscaled expression

SD
Unscaled Expression SEM

Calculated standard error of the mean of the unscaled expression

Corrected Unscaled Expression

Calculated standard error of the mean of the corrected unscaled

SEM

expression

Unscaled Expression(lg)Log2 of the unscaled expression

SD Unscaled Expression(lg)Standard deviation of the unscaled expression (log2)

SEM Unscaled Expression(lg)Standard error of the mean of the unscaled expression (log2)

Expression

Normalized gene expression

254 | CFX Maestro Dx SE SoftwareGraphing

Table 34. Information in the bar chart spreadsheet with Show Details selected, continued

Information

Description

Corrected Expression SD

Calculated standard deviation of the corrected expression

Expression SEM

Standard error of the mean of the expression

Corrected Expression SEM

Calculated standard error of the mean of the corrected expression

Expression(lg)Log2 of the expression (normalized expression) that is used forstatistical analysis

SD Expression(lg)Standard deviation of the expression (log2)

SEM Expression(lg)Standard error of the mean of the expression (log2)

Mean Cq

Mean of the quantification cycle

Cq SD

Standard deviation of the quantification cycle

Cq SEM

Standard error of the mean of the quantification cycle

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Chapter 12 Gene Expression Analysis

Clustergram
The clustergram displays the data in a hierarchy based on the degree of similarity of expression fordifferent targets and samples.
Note: You must choose a reference target to display any of the data plots other than relative
expression for bar charts.
The clustergram image depicts relative expression of a sample or target as follows:n

Upregulation (red) — higher expressionn

Downregulation (green or blue) — lower expressionn

No regulation (black)n

No value calculated (black with a white X)

The lighter the shade of color, the greater the relative expression difference. If no normalized Cq value canbe calculated, the square will be black with a white X.
On the outer edges of the data plot is a dendrogram, which indicates the clustering hierarchy. Targets or
samples that have similar expression patterns will have adjacent branches while those with dissimilar
patterns will be more distant.

Settings
You can set the following options:nCluster By — choose from Targets, Samples, Both, or None.

nSize — adjusts the image size and changes the degree of chart magnification.

nSplit Out Replicates — displays values for the individual replicates.

Tip: You can change the color scheme for from the default Red/Green to Red/Blue by selecting this option
from the right-click menu on of these charts.

Right-Click Menu Options
Right-click menu options for the clustergram are the same as those for the bar chart. See Table 32 on
page 252 for available options. In addition, select Color Scheme to change the downregulation expression
from the default Red/Green to Red/Blue on the chart.

Data Spreadsheet
The spreadsheet displays values for the target, sample, and normalized expression.

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Scatter Plot

Scatter Plot
The scatter plot displays the normalized expression of targets for a control versus an experiment sample. Thelines in the plot indicate the fold change threshold. Data points between the lines indicate that the difference in
expression for that target (gene) is negligible between the samples. Data points outside the lines exceed the foldchange threshold and might be of interest.
The plot image shows the following changes in target expression based on the fold change threshold:nUpregulation (red circle) — relatively higher expressionn

Downregulation (green or blue circle) — relatively lower expressionn

No change (black circle)

Click and drag either threshold line to adjust the fold change threshold value.

Settings
You can set the following options:nControl SamplenExperimental SamplenFold Change Threshold. As you increase or decrease the fold change value, the threshold lines in theplot move accordingly.

Right-Click Menu Options
Right-click menu options for the scatter plot are the same as those for the bar chart. See Table 32 on page
252 for available options. In addition, select Symbol to change the symbol used on the plot from thedefault circle to one of the following:n

Triangle

n

Cross

n

SquarenDiamond

Data Spreadsheet
The spreadsheet displays the values for the target and normalized expression for control andexperimental samples. It also indicates whether targets are up- or downregulated compared to the target
regulation.

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Chapter 12 Gene Expression Analysis

Results Spreadsheet
The Results spreadsheet summarizes the data from all the charts. Table 35 defines the data displayed in
the Results spreadsheet.
Table 35. Information in the Results tab

Information

Description

TargetTarget name (amplified gene)

SampleSample name

Mean Cq

Mean of the quantification cycle

Mean Efficiency Corrected Cq

Mean of the quantification cycle after adjusting for the reaction
efficiency

Normalized Expression

Target expression normalized to a reference target (ΔΔCq)

Relative Normalized Expression

Normalized expression relative to a control sample; also called Fold
ChangeRegulation

Change in expression relative to a control sample

Compared to Regulation

Up- or downregulation of an experimental sample based on theThresholdthreshold setting

Note: Data for replicates are found only in the spreadsheets of data analysis tabs in which Split Out
Replicates has been selected (that is, Clustergram). There might be a discrepancy between
expression data in the gene expression analysis spreadsheets if you select “none” as the control
sample on the bar chart.

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Gene Study

Gene Study
Create a gene study to compare gene expression data from one or more real-time PCR experiments
using an inter-run calibrator to normalize between the experiments. Create a gene study by adding datafrom one or more data files (.pcrd extension) to the gene study. The software groups them into a single file(.mgxd extension).
Note: The maximum number of samples you can analyze in a gene study is limited by the size of thecomputer’s RAM and virtual memory.

Inter-Run Calibration
Inter-run calibration is automatically attempted in every gene study for each target to normalize inter-run
variations between targets assayed in separate real-time PCR runs (that is, different .pcrd files generated
from different plates).
For the software to recognize a sample as an inter-run calibrator, it must share the same target name,sample name, and, if used, biological set name across every plate being compared.
Note: At least one inter-run calibrator sample must be present in the gene study for inter-run
calibration to occur. Targets without appropriate inter-run calibrator samples will be processed
without correction in the gene study (not recommended).
Inter-run calibrators can be applied in two ways:n

Per target — different PCR primers can have different efficiencies. By default the inter-run calibrator
is applied to all of the wells on the same plate that have the same target name, for example the Cq
generated with the same assay.

nEntire study — one inter-run calibrator is selected by the user and applied to the entire gene study.

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Chapter 12 Gene Expression Analysis

Gene Study Dialog Box

The Gene Study dialog box includes two tabs:nStudy Setup tab — manages the runs in the gene study.
Important: Adding or removing data files in a gene study does not change the data in theoriginal file.

nStudy Analysis tab — displays the gene expression data for the combined runs.

Study Setup Tab
Table 36 defines the data that appear in the Study Setup tab.
Table 36. Study Setup tab in the Gene Study dialog box

Column Title

Description

File Name

Name of the run data file (.pcrd extension)

File Folder

Directory that stores the data file for each run in the gene study

Date Created

Date the run data were collected

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Gene Study

Table 36. Study Setup tab in the Gene Study dialog box, continued

Column Title

Description

Well Group Name

Name of the well group that was selected when the file was added to
the gene study
Tip: To analyze one well group in the gene study, you must selectthat well group in the Data Analysis window before importing thedata file into the gene study.

Step

Protocol step that includes the plate read to collect real-time PCR data

Run Type

Either user-defined or PrimePCR run

Protocol Edited

If selected, indicates that the protocol used for a PrimePCR run waseditedView Plate

Opens a plate map of the plate with the data in each of the runs
included in the Gene Study

Preparing a Gene Study
To prepare a gene study
1.

Before importing data into a gene study, do the following in the Data Analysis window:nVerify that samples containing the same content have the same name. In a gene study, thesoftware assumes that wells with the same Target or Sample name contain the same samples.

nAdjust the baseline and threshold (Cq) in the Quantification tab to optimize the data in each run.

n

Select the well group you want to include in the gene study.
In order to show data from one well group in the gene study, that group must be selected before
importing the data file.

The Study Setup tab shows a list of all the runs in the gene study.
2.

In the Gene Study dialog box, choose the Study Setup tab.

3.

Click Add Data Files to select a file from a browser window.
Tip: To quickly add runs to a gene study, drag the data files (.pcrd extension) into the Study
Setup dialog box.

4.

CFX Maestro Dx SE automatically performs the gene study analysis as you add data files. Choose
the Study Analysis tab to view the results.

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Chapter 12 Gene Expression Analysis

To remove runs from the gene studyu

Select one or more files in the list and click Remove.

To add notes about the gene studyuEnter notes about the files and analysis in the Notes text box.

Study Analysis Tab
The Study Analysis tab displays the data from all runs in the gene study. The gene expression data
analysis options are the same as those for a single data file with the following exceptions:n

For bar charts, inter-run calibration values (if calculated) appear when you click Inter-run Calibration.
Note: Only the following sample types can be used as an inter-run calibrator:oUnknownoStandardoPositive Control

Negative control, no template control (NTC), and no reverse transcriptase control (NRT) sample
types cannot be used as an inter-run calibrator.
nn

The Reference Gene Selection tool identifies the tested reference genes and categorizes them as
Ideal, Acceptable, or Unstable based on their stability:o

Ideal reference genes are stable and represent minimal variations across the tested samples.

oAcceptable reference genes are not ideally stable and represent moderate variation acrosstested samples. Use these reference genes in analysis if no Ideal reference genes are present.

oUnstable reference genes represent excessive variation across tested samples. It is
recommended that these genes be excluded from analyses.

The PrimePCR Analysis Controls tool displays the results of the tested samples in a table:o

The Summary tab displays a summary of all tested samples. Samples that passed all of thecontrol assays appear in green. Samples that failed one or more of the control assays appear inyellow.

o

The PCR tab displays the results of the positive PCR control assay. This assay detects inhibition
or experimental problems that affect gene expression.

o

The RT tab displays the results of the reverse transcription control assay. This assay
qualitatively evaluates RT performance and identifies samples where RT performance is likely tocompromise gene expression.

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Gene Studyo

The gDNA tab displays the results of the DNA contamination control assay. This assay
determines whether genomic DNA (gDNA) is present in a sample at a level that might affectqPCR results.

o

The RQ tab displays the results of the RNA quality assays (RQ1 and RQ2). These assays
qualitatively assess whether RNA integrity might adversely affect gene expression.

Gene Study Report Categories
Use the Gene Study Report dialog box to arrange the gene study data into a report. Table 37 lists all theoptions available for a gene study report.
Table 37. Categories for a Gene Study report

Category

OptionDescription

HeaderTitle, subtitle, and logo for the report
Report Information

Date, user name, data file name, data file path,and the selected well group

Gene Study File List

List of all the data files in the Gene Study

Notes

Notes about the data report

Study Analysis: Bar Chart
Analysis Settings

List of the selected analysis parameters

Chart

Gene Expression bar chart showing the data

Target Names

List of targets in the Gene Study

Sample Names

List of samples in the Gene Study

Data

Spreadsheet that shows the data

Target Stability

Target stability data

Inter-run Calibration

Inter-run calibration data

Box-and-Whisker Chart

Gene Expression box-and-whisker chart

Dot-Plot Chart

Gene Expression dot plot chart

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Chapter 12 Gene Expression Analysis

Table 37. Categories for a Gene Study report, continued

Category

OptionDescription

Analysis Settings

Settings for each chart type

Chart

Gene Expression chart showing the data

Data

Spreadsheet listing the data in each target

Study Analysis: ANOVA Data
ANOVA Settings

P-value threshold used in the analysis

ANOVA Results

Table of results from ANOVA and Tukey's HSD
post-hoc analysis

Shapiro-Wilk Normality

Biological group, count, P-value, and any errors

Testthat occur for each target in the analysis

ANOVA Errors

Errors identified during ANOVA calculations

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Gene Study

Creating a Gene Study Report
To create a gene study report
1.

Adjust the gene study report data and charts as needed before creating a report.

2.

Select Tools > Reports in the Gene Study menu to open the Report dialog box.

3.

Choose the options you want to include in the report. The report opens with default options selected.
Select or clear the checkboxes to change whole categories or individual options within a category.
Gene Study Report Categories on page 263 lists the available options to display.

4.

Change the order of categories and items in a report. Drag the options to the required position. Items
can be reordered only within the categories to which they belong.

5.

Click Update Report to update the Report Preview with any changes.

6.

Print or save the report. Click the Print Report button in the toolbar to print the current report. SelectFile > Save to save the report in PDF (Adobe Acrobat Reader file) format and select a location inwhich to save the file. Select File > Save As to save the report with a new name or in a new location.

7.

(Optional) Create a report template with the information you want. To save the current report settings
in a template, select Template > Save or Save As. Then load the report template the next time youwant to make a new report.

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Chapter 12 Gene Expression Analysis

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Appendix A Data Analysis Calculations
CFX Maestro Dx Software, Security Edition calculates formulas automatically and displays the results inthe Data Analysis tabs. This appendix explains in detail how CFX Maestro Dx SE calculates formulas.

Reaction Efficiency
Evidence suggests that using an accurate measure of efficiencies for each primer and probe set will give
you more accurate results when analyzing gene expression data. The default value of efficiency used in
the gene expression calculations is 100%. To evaluate the reaction efficiency, generate a standard curveusing serial dilutions of a representative sample across a relevant dynamic range and then record theefficiency for subsequent gene expression analysis. If your run includes a standard curve, then thesoftware automatically calculates the efficiency and displays it under the Standard Curve on the
Quantification tab when Auto Efficiency is checked in the Targets tab in the Experiment Settings window.
The efficiency (E) in the efficiency formulas refers to the “efficiencies” as described by Pfaffl (2001) and
Vandesompele et al. (2002). In these publications, an efficiency of 2 (perfect doubling with every cycle) is
equivalent to 100% efficiency in this software. You have the option to convert your efficiency calculations
to those used in the software by using the following mathematical relationships:n

E = (% Efficiency * 0.01) + 1n% Efficiency = (E – 1) * 100

Relative Quantity
The formula for relative quantity (ΔCq) for any sample (GOI) is:

Note: This formula is used to calculate Relative Quantity when there is no control sample defined.
Where:n

E = Efficiency of primer and probe set. This efficiency is calculated with the formula(% Efficiency * 0.01) + 1, where 100% efficiency = 2nC q (min) = Average Cq for the Sample with the lowest average Cq for GOI

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Appendix A Data Analysis Calculationsn

C q (sample) = Average Cq for the Samplen

GOI = Gene of interest (one target)

Relative Quantity When a Control Is Selected
When a control sample or biological group is assigned, then the relative quantity (RQ) for any sample witha gene of interest (GOI) is calculated with this formula:

Where:n

E = Efficiency of primer and probe set. This efficiency is calculated with the formula(% Efficiency * 0.01) + 1, where 100% efficiency = 2nCq (control) = Average Cq for the control samplen

Cq (sample) = Average Cq for any samples with a GOIn

GOI = Gene of interest (one target)

Standard Deviation of Relative Quantity
Important: This calculation is applicable only when Analyze Using is set to Samples Only, Sample
Biological Group, or Biological Group Sample.
The formula for standard deviation of the relative quantity is

Where:n

SD Relative Quantity = standard deviation of the relative quantityn

SD CqGOI sample = Standard deviation of the Cq for the sample (GOI)n

Relative Quantity = Relative quantity of the samplen

E = Efficiency of primer and probe set. This efficiency is calculated with the formula(% Efficiency * 0.01) + 1, where 100% efficiency = 2nGOI = Gene of interest (one target)

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Efficiency Corrected Cq (CqE)

Efficiency Corrected Cq (CqE)
The formula for efficiency corrected Cq isWhere:n

E = Efficiency

Mean Efficiency Corrected Cq (MCqE)
The formula for mean efficiency corrected Cq isWhere:n

CqE = Efficiency corrected Cqnn = Number of replicates

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Appendix A Data Analysis Calculations

Normalized Expression
Normalized expression (ΔΔCq) is the relative quantity of your target (gene) normalized to the quantities of
the reference targets (genes or sequences) in your biological system. To select reference targets, open
the Experiment Settings window and click the reference column for each target that serves as a reference
gene.
The formula for normalized expression, which uses the calculated Relative Quantity (RQ) calculation, is

Where:n

RQ = Relative quantity of a samplen

Ref = Reference target in a run that includes one or more reference targets in each samplen

GOI = Gene of interest (one target)

Provided that reference targets do not change their expression level in your biological system, thecalculation of normalized expression will account for loading differences or variations in cell number thatare represented in each of your samples.

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Normalized Expression When a Control Is Selected

Expression and Relative Quantity for Biological Groups
When Analyze Using is set to Biological Groups Only, the software displays the average expression(normalized expression or relative quantity, depending on mode selection) of the samples within the
biological group. Because expression is typically log-normally distributed, the expression is averaged
using the geometric mean:

Where:
nExp1, Exp2, Expn = Relative quantity or normalized expression of the samples in the biological groupnn = Number of samples in the biological group

Normalized Expression When a Control Is Selected
When you select a control sample in the Experiment Settings window, the software sets the expression
level of the control sample to 1. In this situation, the software normalizes the relative quantities of all target(gene) expression to the control quantity (a value of 1). This normalized expression is equivalent to theunscaled normalized expression analysis when a control is chosen.
Note: This is also known as relative normalized expression (RNE) and fold change.

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Appendix A Data Analysis Calculations

Standard Deviation for the Normalized Expression
Rescaling the normalized expression value is accomplished by dividing the standard deviation of thenormalized expression by the normalized expression value for the highest or lowest individual expression
levels, depending on the scaling option you choose. The formula for standard deviation (SD) of thenormalization factor is

Where:n

RQ = Relative quantity of a samplenSD = Standard deviationn

NF = Normalization factorn

Ref = Reference targetnn = Number of reference targets

When a control sample is assigned, you do not need to perform this rescaling function on the standard
deviation, as shown in the following formula:

Where:nNE = Normalized expressionn

RQ = Relative quantity of a samplenSD = Standard deviationn

GOI = Gene of interest (one target)

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Normalized Expression Scaled to Highest Expression Level

Normalized Expression Scaled to Highest Expression Level
When the run does not include controls, scale the normalized expression (NE) for each target (gene) bydividing the expression level of each sample by the highest level of expression in all the samples. The
software sets the highest level of expression to a value of 1 and rescales all the sample expression levels.
The formula for highest scaling is

Where:n

GOI = Gene of interest (target)

Normalized Expression Scaled to Lowest Expression Level
When the run does not include controls, scale the normalized expression (NE) for each target (gene) bydividing the expression level of each sample by the lowest level of expression in all the samples. The
software sets the lowest level of expression to a value of 1 and rescales all the sample expression levels.
The formula for lowest scaling is

Where:n

GOI = Gene of interest (target)

Normalized Expression Scaled to Average Expression Level
When the run does not include controls, scale the normalized expression (NE) for each target (gene) bydividing the expression level of each sample by the geometric mean level of expression of all the samples.
The software sets the average level of expression to a value of 1 and rescales all the sample expression
levels. The formula for average scaling is

Where:n

GOI = Gene of interest (target)n

GM = Geometric mean of normalized expression for all samples

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Appendix A Data Analysis Calculations

Standard Deviation for the Scaled Normalized Expression
Rescaling the scaled normalized expression (NE) value is accomplished by dividing the standard
deviation (SD) of the normalized expression by the normalized expression value for the highest (MAX) or
lowest (MIN) expression level, depending on which scaling option you choose.
Note: When a control sample is assigned, you do not need to perform this rescaling function on thestandard deviation.
The calculation for this formula isWhere:nNE = Normalized expressionnSD = Standard deviationn

GOI = Gene of interest (target)n

MAX = Highest expression leveln

MIN = Lowest expression level

274 | CFX Maestro Dx SE SoftwareError Bars for Standard Deviation(lg) and Standard Error of the Mean (lg)Error Bars for Standard Deviation(lg) and Standard Error of
the Mean (lg)
In addition to the use of confidence intervals, error bars may be displayed for biological groups based on
the standard deviation or standard error of the mean of the log2 of the expression. The error bars arecalculated as follows:
RQ Lower Error Bar = 2RQ(lg)-SD RQ(lg) or 2RQ(lg)-SEM RQ(lg)RQ Upper Error Bar = 2RQ(lg)+SD RQ(lg) or 2RQ(lg)+SEM RQ(lg)
Where:n

RQ(lg) = log2 of the relative quantity for the biological groupn

SD RQ(lg) = standard deviation of the relative quantity (log2)n

SEM RQ(lg) = standard error of the mean of the relative quantity (log2)

Exp. Lower Error Bar = 2Exp.(lg)-SD Exp.(lg) or 2Exp.(lg)-SEM Exp.(lg)Exp. Upper Error Bar = 2Exp.(lg)+SD Exp.(lg) or 2Exp.(lg)+SEM Exp.(lg)
Where:
nExp.(lg) = log2 of the expression (normalized expression) for the biological groupn

SD RQ(lg) = standard deviation of the expression (log2)n

SEM RQ(lg) = standard error of the mean of the expression (log2)

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Appendix A Data Analysis Calculations

Fold Change
Fold change is a measure of the increase or decrease in the expression of a target for an experimental
versus a control sample or biological group and is determined as follows:
If Expression (experimental) > Expression (control):If Expression (experimental) < Expression (control):Note: For Graphing, the Expression is based on either the relative quantity or normalized expression,
depending on the selected mode (see Graphing on page 242). However, for the Scatter Plot andClustergram fold change is always calculated from the normalized expression.

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Corrected Values Formulas

Corrected Values Formulas
Important: These calculations are applicable only when Analyze Using is set to Samples Only,
Sample Biological Group, or Biological Group Sample.
A difference between corrected values and non-corrected values is seen only if a standard curve iscreated as part of the real-time PCR run. The software uses three equations to determine the error
propagation:nStandard ErrornStandard Error for Normalized ExpressionnStandard Error for the Normalized Gene of Interest (target)

The formula for standard error is

Where:
nn = Number of reference targets (genes)n

SD = Standard deviation

The standard error for the normalization factor in the normalized expression formula isWhere:
nn = Number of reference targetsnSE = Standard errorn

NF = Normalization factorn

RQ = Relative quantity

The standard error for normalized gene of interest (GOI) formula isWhere:nSE = Standard errorn

GOI = Gene of interest (one target)

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Appendix A Data Analysis Calculationsn

NF = Normalization factornn = Number of reference targets

Confidence Interval Calculation for Biological Group Analysis
When conducting biological groups analysis (Analyze Using is set to Biological Groups Only), theconfidence intervals are calculated for relative quantity and relative normalized expression.
The confidence intervals are calculated in log-scale based on t-distribution using the following formula:

Where:n= Mean expression of the log-scale expression levels of the samples in the biological groupn

SD = standard deviation of the log-scale expression levels of the samples in the biological groupnn = number of the samples in the biological groupnt = obtained from the t-distribution based on the degrees of freedom and the alpha level
Note: The alpha level can be set using the P-value threshold field in the Graphing tab.

After the confidence intervals are calculated, they are converted to linear scale and presented in the Gene
Expression Data Table and the bar chart in the Graphing tab.

Box and Whisker Chart Calculations
The Box and Whisker chart displays the distribution of the expression values within a biological group by
plotting the data as quartiles. The 1st and 3rd quartiles are represented by the lower and upper bounds ofthe box, respectively. The median is displayed as a solid line across the box. The whiskers represent theminimum and maximum non-outlier values in the data set. Outliers are values that exceed the 1st and 3rdquartiles by 1.5 times the inter-quartile range.
Note: If there is only one sample in the biological group it is shown as a single circle, indicating asingle data point.
The following Box and Whisker chart demonstrates how these data is represented.

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Box and Whisker Chart Calculations

LEGEND1.

Outlier. This outlier's value is > Q3 + (1.5 x [Q3 - Q1]).
Note: Place the cursor over the circle to view a tooltip that displays the sample name andthe relative quantity or normalized expression information depending on which mode isselected.

2.

Maximum non-outlier demarcation

3.

Upper/3rd quartile (Q3). 75% of the expression values are less than Q3.

4.

Median, or middle-most value, of the rank-ordered expression values

5.

Lower/1st quartile (Q1). 25% of the expression values are less than Q1.

6.

Minimum non-outlier demarcation

7.

Outlier. This outlier's value is < Q1 - (1.5 x [Q3 - Q1]).

User Guide | 279

Appendix A Data Analysis Calculations

280 | CFX Maestro Dx SE Software

Appendix B Audit Trails
CFX Maestro Dx Software, Security Edition creates audit trails for data and gene study files (.prcd and.mgxd files respectively). Any changes made to or actions performed on secure data and gene study files
are captured in the file’s audit trail when the file is saved. CFX Maestro Dx SE creates a separate audit
trail for each file.
You can choose File > Save As and save secure signed or unsigned data and gene study files intoanother folder or with another name. The new file inherits the audit trail from the original file. The audit trail
for the new file also includes the Save As activity. Changes to or actions performed on the new file arecaptured in its own audit trail. The original file retains its audit trail, in which further activity is captured.
Auditable Events on page 283 lists the auditable events that the software captures.

Viewing Audit Trails
Each audit trail displays the following information:nnAudit header detailoFile version — the saved version of the fileoDate — the date of the current auditable evento

User — the Windows domain and user name of the logged in useroComment — the last saved commento

Signature — the electronic signature of the last person who signed the fileo

Signature reason — the reason for the signatureoApplication — CFX Maestro Dx SEoApplication version — the current version of CFX Maestro Dx SE

oFull user — the full name of the logged-in useroMachine — the computer on which CFX Maestro Dx SE is installed

Audit change detailoObject — the item that was changed (the audited item)

User Guide | 281

Appendix B Audit Trailso

Old value — the previous valueo

New value — the new valueoDescription — the description of the change

To view the audit trailu

In the open data or gene study file, select View > Audit Trail. The file's audit trail appears.

By default, the data are sorted by date and time, and all events appear in the expanded view. You canfilter the view by user name and object, and collapse the expanded view to easily sort by any header
field. You can also view the audit trail as an html report.

To sort by user nameuSelect the target user from the User Name dropdown list.

To sort by object
u

Select the target from the Object dropdown list.

To hide the full description of eventsuClick Collapse All.

To sort data in the change detail tableu

Click the diamond symbol in the data column header to perform an ascending sort (A to Z, smallest
number to largest, or earliest to most recent).

282 | CFX Maestro Dx SE Software

Auditable Events

To print the audit trail
1.

Click HTML Report to display the audit trail in a web browser.

2.

In your browser window, do one of the following:nSelect File > Print.

nRight-click the report and select Print.

Auditable Events
CFX Maestro Dx SE captures the following auditable events in data and gene study files.

Auditable events during runn

Run Start Timen

Run Time Plate editsn

Run Time Protocol editsn

Run End Time

Auditable events when a data file is creatednData file creatednInterpolated Plate Reads added by the system

Auditable events when a data file is savednGeneraloNameoSigningoPlate Setupo

Display WellsoAnalyzed fluorophoresoPlate editsoAnalysis modeo

PCR Active Well Group

User Guide | 283

Appendix B Audit Trails

n

n

n

n

n

Quantification taboActive stepoSettings — Cq Determination modeoSettings — Baseline SettingoDrift correction appliedoSettings — Cycles to AnalyzeoSettings — Analysis ModeoSettings — Baseline Threshold

Melt Curve taboActive stepoPeak type displayedoPeak analysis threshold

End Point taboActive fluorophore/targeto

End cycles to averageoTolerance calculation methodoPercentage of range

Allelic Discrimination taboX- and Y-axis fluorophoreoSelect cycle numberoView call map

Gene Expression tab — All plotsoExperiment Settings — Target referenceoExperiment Settings — Sample controloExperiment Settings — Auto efficiencyoExperiment Settings — Efficiency

284 | CFX Maestro Dx SE Software

Auditable Events

n

n

n

n

Gene Expression tab — GraphingoAnalysis modeoGraph dataoX-axisoY-axiso

Scaling optionoError baroError bar multiplieroP-value threshold

Gene Expression tab — Clustergramo

Cluster ByoSplit out replicates

Gene Expression tab — Scatter PlotoControl biological groupoExperimental biological groupo

Fold change threshold

Gene Expression tab — ANOVAonnP-value threshold

Plate Setup — View/Edit Plateo

Settings — PlateTypeo

Settings — Unitso

Editing Tools — Flip PlateoWell groupsoPlate fluorophores

Plate Setup — Replace Plate and Apply PrimePCR FileoPlate Setup Import

User Guide | 285

Appendix B Audit Trails

Audit Changes for Gene Study Files
GeneralnNamenStudy Setup tabon

Add/Remove data files

Study Analysis tab

286 | CFX Maestro Dx SE Software

Appendix C LIMS Integration
You can configure CFX Maestro Dx Software, Security Edition for use with a laboratory information
management system (LIMS). For LIMS integration, CFX Maestro Dx SE requires plate setup information
generated by the LIMS platform (a LIMS file, *.plrn), a protocol file created using CFX Maestro Dx SE(*.prcl), a defined data export location, and a defined export format.
After the run is finished, CFX Maestro Dx SE generates a data (.pcrd) file and saves it to a defined data
export folder location. CFX Maestro Dx SE can also create a LIMS-compatible data file in .csv format andsave it to the same location.

Creating LIMS-Compatible Data Files
This appendix explains how to set up CFX Maestro Dx SE to create, save, and export LIMS-compatible
data files.

Setting Up LIMS Folder and Data Export Options
By default, CFX Maestro Dx SE saves the LIMS protocols, files, and data export files to this folder:
C:\Users\Public\Documents\Bio-Rad\CFX_Dx\LIMS
You can configure CFX Maestro Dx SE to save the files to another folder and change the export options
for LIMS data.

To set up a LIMS folder and data export options
1.

On the Home window, select Tools > Options.

2.

In the Options dialog box, select Data Export Settings.

User Guide | 287

Appendix C LIMS Integration

3.

(Optional) Select Automatically export LIMS data at end of run.
The software will automatically export LIMS data after each run and save it to the specified location.

4.

To change the default export options for LIMS data, click LIMS Data Export Settings.
Important: Only LIMS data exported as a .csv file can be imported back into CFX Maestro Dx
SE.

5.

In the LIMS Data Export Format Settings dialog box, select the required export options and click OK.

6.

In the Options dialog box, navigate to and select a default folder in which you want to save the LIMS
data files. You can select a different location for each file type:7.

nProtocoln

LIMS filenData export

Click OK to save the changes and close the Options dialog box.

288 | CFX Maestro Dx SE Software

Creating LIMS-Compatible Data Files

Creating a LIMS Protocol
To start a LIMS run, create a CFX Maestro Dx SE protocol file (*.prcl) and save it in the designated LIMS
protocol folder location.
See Chapter 7, Creating Protocols for more information.

Creating a LIMS File
A LIMS file (*.plrn) contains the plate setup details and the protocol file name. This file is generated byyour internal LIMS. CFX Maestro Dx SE uses the LIMS file to create a plate file to use with a protocol file.
CFX Maestro Dx SE provides plate import template files that you can edit to create custom LIMS platefiles.
Tip: This task should be performed by a LIMS specialist.

To create a LIMS file1.

In the Home window, select View > Show > LIMS File Folder.

2.

Open the LIMS Templates folder and select a .csv file to import into your internal LIMS.

3.

Edit the template file by filling in the required fields listed in Table 38.

4.

Do one of the following:n

To save your changes for future use, save the file as a .csv file.

n

To save your changes and use the file immediately, save the file with the .plrn extension.

n

Save the template with the file name extension .plrn to the LIMS File folder.

Important: CFX Maestro Dx SE can open only the .plrn file. You must save the .csv file as .plrn
in order to start the LIMS run.
Table 38. Definition of LIMS .csv file contents

ColumnRow

Description

Content

PurposeA

1

Plate Header

Do not edit

Predefined

A,B,C

2

Field/Data/Instruction

Do not edit

PredefinedB

3

Version

Do not edit

PredefinedB

4Plate Size

Do not edit

PredefinedUser Guide | 289

Appendix C LIMS Integration

Table 38. Definition of LIMS .csv file contents, continued

ColumnB

Row
5

Description

Content

PurposePlate TypeEnter “BR White,” “BR

Required

Clear,” or other calibrated
plate typeB

6

Scan Mode

Enter “SYBR/FAM Only:,”

Required“All Channels,” or “FRET”
B

7

Units

Enter one of the following

Required“copy number,” “folddilution,” “micromoles,”“nanomoles,”“picomoles,”“femtomoles,”“attomoles,” “milligrams,”“micrograms,”“nanograms,”“picograms,”“femtograms,”“attograms,” or “percent”
B

8

Run IDEnter short description or

Optional

bar code identifying thisrun (30-character
maximum, commas notallowed)B

9

Run Notes

Enter run description

Optional

B

10

Run Protocol

Enter protocol file name

Requiredexactly as listed.
A

11

Data FileEnter data file name

Optional

A

12-15

TBD/Empty

Do not edit

PredefinedA

16

Plate Data

Do not edit

PredefinedA

17-113Well Position

Do not edit

Predefined

290 | CFX Maestro Dx SE Software

Creating LIMS-Compatible Data Files

Table 38. Definition of LIMS .csv file contents, continued

ColumnB-G

Row

Description

Content

PurposeCh1 Dye, Ch2 Dye, Ch3

Enter one calibrated dye

RequiredDye, Ch4 Dye, Ch5 Dye,name (for example,

FRET“FAM”) for each channel
being used

H

Sample TypeEnter one of the following

Requiredsample types “Unknown,”“Standard,” “Positive
Control,” “Negative
Control,” “NTC,” or “NRT”
I

Sample NameEnter sample name

Optional

J-O

CH1 Target, CH2 Target,Enter target name for

Optional

CH3 Target, CH4 Target,each channel used

CH5 Target, FRET
TargetP

Collection NameEnter biological set name

Optional

Q

ReplicateEnter a positive integer

Optional

for each set of replicates.
The value cannot be
zero.
R-W

CH1 Quantity, CH2

Enter quantity values for

Required for

Quantity, CH3 Quantity,

any standards. Enter

all standards

CH4 Quantity, CH5concentration in decimal

Quantity, FRET Quantity

form.

User Guide | 291

Appendix C LIMS Integration

Table 38. Definition of LIMS .csv file contents, continued

ColumnRow

X

Description

Content

PurposeWell Note

Enter well note (20-

Optional

character maximum)
Note: Although
CFX Maestro Dx SE
has a 20-character
limit when enteringnotes in the Well
Note via thesoftware, the Well
Note field cancontain up to 500
characters if
included in an
imported .plrn file.
However,
CFX Maestro Dx SE
will display only the
first 20 characters.
The exported .pcrd
file will contain all
characters in the
Well Note field, nodata is lost.
Y-AD

Ch1 Well Color, Ch2 Well

Enter any user-definedColor, Ch3 Well Color,

trace style color in a 32 bit

Ch4 Well Color, Ch5 Well

integer (argb) decimal

Color, FRET Well Color

formatOptional

Starting a LIMS Run
To start a LIMS run
1.

Do one of the following to open a LIMS .plrn file:n

In the Home window, select View > Show > LIMS File Folder and open the target .plrn file.

n

In the Home window, select File > Open > LIMS File and open the target .plrn file.

The file opens in the Start Run tab in the Run Setup wizard. The Start Run tab displays information
about the experiment to run. It also displays the connected instrument block or blocks on which you

292 | CFX Maestro Dx SE Software

Creating LIMS-Compatible Data Files

can run the experiment.
2.

In the Start Run tab, select an instrument and click Start Run.

Exporting Data to a LIMS
When the run completes, CFX Maestro Dx SE generates a data (.pcrd) file and saves it to the defined data
export folder location.

To export the data file to a LIMS
uOpen the .pcrd file and select Export > Export to LIMS Folder.

Tip: If you select Automatically Export Data after Run in LIMS Options, CFX Maestro Dx SE createsa LIMS-compatible data file in .csv format and saves it in the same folder.

User Guide | 293

Appendix C LIMS Integration

294 | CFX Maestro Dx SE Software

Appendix D Troubleshooting CFX Maestro Dx
Software, Security Edition
This appendix provides suggestions for troubleshooting issues you might encounter while upgrading or
running CFX Maestro Dx Software, Security Edition.

Whitelist CFX Maestro Dx Software, Security Edition Files andFolders
In order to protect against viruses and malware, your IT department might have implemented very tightsoftware security measures. These measures might impact the time to upgrade or run CFX Maestro Dx
SE.
To improve the performance of CFX Maestro Dx SE, Bio-Rad recommends that your IT department
whitelist the following files and folders in the Firewall settings in your antivirus software installed on the
CFX Maestro Dx SE computer:

FoldersnC:\Program Files (x86)\Bio-Rad\CFX_MDx

n

C:\ProgramData\Bio-Rad\CFX_MDx

n

C:\Users\Public\Documents\Bio-Rad\CFX_MDx

Files
n

All .exe files located in the folder C:\Program Files (x86)\Bio-Rad\CFX_MDxnR.exe and Rscript.exe (located in the folder C:\Program Files (x86)\Bio-Rad\CFX_MDx\ R\
R-3.3.1\bin)User Guide | 295

Appendix D Troubleshooting CFX Maestro Dx Software, Security Edition

Application Log
Before starting a new run, the CFX Opus Dx system initiates a self-diagnostic test to verify that it isrunning within specifications. The software records results of this test in the Run Log and Application Log
file. If you notice a problem in one or more experiments, open the run and application logs to find out when
the problem started.
CFX Maestro Dx SE Dx tracks information about the state of an instrument during a run in the Application
Log. Use these logs to track events that occur on instruments and in the software and for troubleshooting.

To open the Application logu

In the Home window, select View > Application Log.

To view the Application Log as an HTML file, click the HTML Report button.

296 | CFX Maestro Dx SE Software

Troubleshooting

Retrieving Application and Firmware Log Files
The application and firmware logs contain information detailing actions performed during the use of thesoftware and performance of runs. These logs also record any software or firmware errors that occur
during operation of the software or instrument.

To access the application and firmware log files:
1.

In the Detected Instruments pane, right-click on the instrument.

2.

Select Retrieve Log Files.

3.

In the Browse for Folder dialog, select the destination folder on your network or a local drive to which
you want to save the log files.
Note: The folder is titled "Logs."

4.

Click OK to save the files.
Important: Saving a log file with the same file name as an existing log file will overwrite theexisting log file.

Troubleshooting
Typically, software and instrument communication problems can be resolved by restarting your computer
and the system. Be sure to save any work in progress before restarting.
Note: Verify that your computer has sufficient RAM and free disk space. The minimum RAM is 4 GB
and the minimum hard disk space is 128 GB.

Power Failure
In a power failure, the instrument and computer will shut down. If the power failure is short, the instrument
will resume running a protocol but the Application Log will note the power failure. Depending on thecomputer settings and the length of time the power is off, the instrument and software attempt to continue
running depending on the protocol step:n

If the protocol is in a step with no plate read, the protocol continues running as soon as the instrument
regains power.

n

If the protocol is in a step with a plate read, the instrument waits for the software to restart and resumecommunication to collect the data. In this situation, the protocol continues only if the software is notshut down by the computer. When the computer and software start up again, the protocol continues.

User Guide | 297

Appendix D Troubleshooting CFX Maestro Dx Software, Security Edition

Transferring Files to the CFX Maestro Dx SE Computer
You can transfer data and log files located on the instrument to the hard drive of an attached CFX Maestro
Dx SE computer.
Tip: All files in the real-time data folder on the instrument base are transferred to the computer.
Note: From CFX Opus Dx instruments, you can transfer only log files. All log files on the instrument
are transferred to the computer.

To retrieve files from the instrument
1.

In the Detected Instruments pane on the Home window, right-click the target instrument and selectRetrieve Log Files.

2.

Choose a folder location to save the retrieved files.

3.

Click OK.

Installing CFX Maestro Dx Software, Security Edition Manually
To manually install CFX Maestro Dx SE
1.

If necessary, disconnect any connected instruments from the computer.
Locate and disconnect the instrument’s USB cable on the CFX Maestro Dx SE computer. The end
inserted in the instrument can remain in place.

2.

Log in to the CFX Maestro Dx SE computer with administrative privileges.

3.

Insert the CFX Maestro Dx SE USB drive into the computer’s USB port.

4.

In Windows Explorer, navigate to and open the CFX Maestro Dx SE USB drive.

5.

Open the CFX folder and double-click CFXMaestroDxSetup.exe to install CFX Maestro Dx SE.

6.

Follow the instructions on the screen to install the software.
When completed, the Bio-Rad CFX Maestro Dx Software, Security Edition splash screen appears on
the computer screen and the Bio-Rad CFX Maestro Dx Software, Security Edition icon appears on
the desktop.

7.

Safely eject the software USB drive and start CFX Maestro Dx SE.

Reinstalling the Drivers
To reinstall the instrument driversu

In the Home window, select Tools > Reinstall Instrument Drivers.

298 | CFX Maestro Dx SE Software

Troubleshooting

Note: If you have problems with the software communicating with a real-time system after you
reinstall the drivers and check USB connection, contact Bio-Rad Technical Support.

User Guide | 299

Appendix D Troubleshooting CFX Maestro Dx Software, Security Edition

300 | CFX Maestro Dx SE Software

Appendix E Bio-Rad Free and Open-Source
Notices for PCR ProductsThis document includes licensing information relating to free, open-source, and public-source softwareand data (together, the “MATERIALS”) included with or used to develop Bio-Rad products and services.
The terms of the applicable free, open-source, and public-source licenses (each an “OPEN LICENSE”)
govern Bio-Rad’s distribution and your use of the MATERIALS. Bio-Rad and the third-party authors,licensors, and distributors of the MATERIALS disclaim all warranties and liability arising from all use anddistribution of the MATERIALS. To the extent the OSS is provided under an agreement with Bio-Rad thatdiffers from the applicable OSS LICENSE, those terms are offered by Bio-Rad alone.
Bio-Rad has reproduced below copyright and other licensing notices appearing within the MATERIALS.
While Bio- Rad seeks to provide complete and accurate copyright and licensing information for all
MATERIALS, Bio-Rad does not represent or warrant that the following information is complete, correct, or
error-free. MATERIALS recipients are encouraged to (a) investigate the identified MATERIALS to confirm
the accuracy of the licensing information provided and (b) notify Bio-Rad of any inaccuracies or errors
found in this document so that Bio-Rad may update this document accordingly.
Certain OPEN LICENSES (such as the Affero General Public Licenses, Common Development andDistribution Licenses, Common Public License, Creative Commons Share-Alike License, Eclipse Public
License, Mozilla Public Licenses, GNU General Public Licenses, GNU Library/Lesser General Public
Licenses, and Open Data Commons Open Database License) require that the source materials be madeavailable to recipients or other requestors under the terms of the same OPEN LICENSE.
The corresponding open source software is available for download from the links in the section that
follows.

User Guide | 301

Appendix E Bio-Rad Free and Open-Source Notices for PCR Products

Software Notices
ZedGraph
Project homepage/download site:
https://sourceforge.net/projects/zedgraph/
Bio-Rad source code site:
https://github.com/bio-rad-lsg-open-source/ZedGraph-5.0.1
External source code site:
https://github.com/ZedGraph/ZedGraph
Project licensing notices:/LICENSE-LGPL.txt:
See LGPL-2.1 in the Standard OSS License Text appendix to this document.
/sources/ZedGraph/LICENSE-LGPL.txt:
See LGPL-2.1 in the Standard OSS License Text appendix to this document.

Standard Open License Text
LGPL-2.1
GNU LESSER GENERAL PUBLIC LICENSE
Version 2.1, February 1999
Copyright (C) 1991, 1999 Free Software Foundation, Inc. 59 Temple
Place, Suite 330, Boston, MA 02111-1307 USA Everyone is permitted tocopy and distribute verbatim copies of this license document, butchanging it is not allowed.
[This is the first released version of the Lesser GPL. It also counts
as the successor of the GNU Library Public License, version 2, hence
the version number 2.1.]

302 | CFX Maestro Dx SE Software

Standard Open License Text

Preamble
The licenses for most software are designed to take away your freedom
to share and change it. By contrast, the GNU General Public Licenses
are intended to guarantee your freedom to share and change free
software--to make sure the software is free for all its users.
This license, the Lesser General Public License, applies to somespecially designated software packages--typically libraries--of theFree Software Foundation and other authors who decide to use it. You
can use it too, but we suggest you first think carefully about
whether this license or the ordinary General Public License is thebetter strategy to use in any particular case, based on the
explanations below.
When we speak of free software, we are referring to freedom of use,
not price. Our General Public Licenses are designed to make sure thatyou have the freedom to distribute copies of free software (andcharge for this service if you wish); that you receive source code or
can get it if you want it; that you can change the software and use
pieces of it in new free programs; and that you are informed that you
can do these things.
To protect your rights, we need to make restrictions that forbid
distributors to deny you these rights or to ask you to surrendertheserights.
These
restrictionstranslatetocertain
responsibilities for you if you distribute copies of the library or
if you modify it.
For example, if you distribute copies of the library, whether gratis
or for a fee, you must give the recipients all the rights that we
gave you. You must make sure that they, too, receive or can get thesource code. If you link other code with the library, you must
provide complete object files to the recipients, so that they canrelink them with the library after making changes to the library andrecompiling it. And you must show them these terms so they know theirrights.
We protect your rights with a two-step method: (1) we copyright thelibrary, and (2) we offer you this license, which gives you legal
permission to copy, distribute and/or modify the library.
To protect each distributor, we want to make it very clear that there
is no warranty for the free library. Also, if the library is modified
by someone else and passed on, the recipients should know that what
they have is not the original version, so that the original author'sUser Guide | 303

Appendix E Bio-Rad Free and Open-Source Notices for PCR Products

reputation will not be affected by problems that might be introducedby others.
Finally, software patents pose a constant threat to the existence ofany free program. We wish to make sure that a company cannot
effectively restrict the users of a free program by obtaining a
restrictive license from a patent holder. Therefore, we insist thatany patent license obtained for a version of the library must be
consistent with the full freedom of use specified in this license.
Most GNU software, including some libraries, is covered by theordinary GNU General Public License. This license, the GNU Lesser
General Public License, applies to certain designated libraries, andis quite different from the ordinary General Public License. We use
this license for certain libraries in order to permit linking thoselibraries into non-free programs.
When a program is linked with a library, whether statically or usinga shared library, the combination of the two is legally speaking a
combined work, a derivative of the original library. The ordinary
General Public License therefore permits such linking only if theentire combination fits its criteria of freedom. The Lesser General
Public License permits more lax criteria for linking other code withthe library.
We call this license the "Lesser" General Public License because it
does Less to protect the user's freedom than the ordinary General
Public License. It also provides other free software developers Lessof an advantage over competing non-free programs. These disadvantages
are the reason we use the ordinary General Public License for many
libraries. However, the Lesser license provides advantages in certain
special circumstances.
For example, on rare occasions, there may be a special need toencourage the widest possible use of a certain library, so that it
becomes a de-facto standard. To achieve this, non-free programs mustbe allowed to use the library. A more frequent case is that a freelibrary does the same job as widely used non-free libraries. In thiscase, there is little to gain by limiting the free library to free
software only, so we use the Lesser General Public License.
In other cases, permission to use a particular library in non-freeprograms enables a greater number of people to use a large body offree software. For example, permission to use the GNU C Library innon- free programs enables many more people to use the whole GNU

304 | CFX Maestro Dx SE Software

Standard Open License Text

operating system, as well as its variant, the GNU/Linux operating
system.
Although the Lesser General Public License is Less protective of theusers' freedom, it does ensure that the user of a program that is
linked with the Library has the freedom and the wherewithal to run
that program using a modified version of the Library.
The precise terms and conditions for copying, distribution andmodification follow. Pay close attention to the difference between a"work based on the library" and a "work that uses the library". The
former contains code derived from the library, whereas the lattermust be combined with the library in order to run.
GNU LESSER GENERAL PUBLIC LICENSETERMS AND CONDITIONS FOR COPYING, DISTRIBUTION AND MODIFICATION
0. This License Agreement applies to any software library or otherprogram which contains a notice placed by the copyright holder orother authorized party saying it may be distributed under the termsof this Lesser General Public License (also called "this License").
Each licensee is addressed as "you".
A "library" means a collection of software functions and/or dataprepared so as to be conveniently linked with application programs(which use some of those functions and data) to form executables.
The "Library", below, refers to any such software library or work
which has been distributed under these terms. A "work based on the
Library" means either the Library or any derivative work undercopyright law: that is to say, a work containing the Library or a
portion of it, either verbatim or with modifications and/ortranslated straightforwardly into another language. (Hereinafter,translationisincluded
without
limitation
in
the
term
"modification".)"Source code" for a work means the preferred form of the work formaking modifications to it. For a library, complete source code means
all the source code for all modules it contains, plus any associatedinterface definition files, plus the scripts used to control
compilation and installation of the library.
Activities other than copying, distribution and modification are notcovered by this License; they are outside its scope. The act ofrunning a program using the Library is not restricted, and output

User Guide | 305

Appendix E Bio-Rad Free and Open-Source Notices for PCR Products

from such a program is covered only if its contents constitute a work
based on the Library (independent of the use of the Library in a tool
for writing it). Whether that is true depends on what the Library
does and what the program that uses the Library does.
1. You may copy and distribute verbatim copies of the Library's
complete source code as you receive it, in any medium, provided thatyou conspicuously and appropriately publish on each copy anappropriate copyright notice and disclaimer of warranty; keep intact
all the notices that refer to this License and to the absence of any
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2. You may modify your copy or copies of the Library or any portionof it, thus forming a work based on the Library, and copy anddistribute such modifications or work under the terms of Section 1
above, provided that you also meet all of these conditions:a) The modified work must itself be a software library.
b) You must cause the files modified to carry prominent notices
stating that you changed the files and the date of any change.
c) You must cause the whole of the work to be licensed at no charge
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Appendix E Bio-Rad Free and Open-Source Notices for PCR Productsa) Accompany the combined library with a copy of the same work basedon the Library, uncombined with any other library facilities. This
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Appendix F References1.

Sugimoto et al. (1996). Improved thermodynamic parameters and helix initiation factor to predictstability of DNA duplexes. Nucleic Acids Research 24, 4,501–4,505.

2.

Breslauer KJ et al. (1986). Predicting DNA duplex stability from the base sequence. Proc Nat Acad
Sci 83, 3,746–3,750.

3.

Hellemans J et al. (2007). qBase relative quantification framework and software for management andautomated analysis of real-time quantitative PCR data. Genome Biol 8, R19.

4.

Livak JL et al. (1995). Towards fully automated genome-wide polymorphism screening. Nature
Genetics 9, 341–342.

5.

Pfaffl MW (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic
Acids Research 29, 2,002–2,007.

6.

Vandesompele J et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by
geometric averaging of multiple internal control genes. Genome Biology 3, 1–12.

7.

Fox J (2008). Applied Regression Analysis and Generalized Linear Models. 2nd ed (New York:SAGE Publications, Inc.).

Minpack Copyright Notice (1999) University of Chicago. All rights reserved
Redistribution and use in source and binary forms, with or without modification, are permitted provided
that the following conditions are met:
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Redistributions of source code must retain the above copyright notice, this list of conditions, and thefollowing disclaimer.

2.

Redistributions in binary form must reproduce the above copyright notice, this list of conditions, andthe following disclaimer in the documentation and/or other materials provided with the distribution.

3.

The end-user documentation included with the redistribution, if any, must include the following
acknowledgment:“This product includes software developed by the University of Chicago, as Operator of Argonne National
Laboratory.”

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