Amplification: Consumables

Amplification Reagents and Plastics

PCR AND GENE EXPRESSION WORKFLOW

2

1

Pages 2–5
SingleShot Cell Lysis RT-qPCR Kits
■ One-Step and Two-Step
SYBR® Green or Probes Formats

CELL LYSIS■

Pages 6–9
RNA Isolation Kits Selection Guide
Aurum Sample Preparation Kits and
PureZOL RNA Isolation Reagent■ Ordering Information■

RNA
ISOLATION■

4

Pages 16–35

Real-Time PCR Reagents Selection Guide
SsoAdvanced Universal Supermixes■ iTaq Universal Supermixes■ Reliance One-Step Multiplex RT-qPCR Supermix■ iTaq Universal One-Step Kits
■ iQ Supermixes■ Precision Melt Supermix■ Precision Blue Real-Time PCR Dye■ High-Fidelity and Standard PCR Reagents■ Real-Time PCR Reagent Compatibility
with Instruments■ Ordering Information■■

REVERSE
TRANSCRIPTION

3

Pages 10–15
Reverse Transcription Kit Selector
Reliance Select cDNA Synthesis Kit■ iScript cDNA Synthesis Kits
■ Ordering Information■■

PCR AND REAL-TIMEPCR REAGENTS

5

6

Pages 36–39

PREAMPLIFICATION

Preamplification
Selection Guide■ iScript Explore
One-Step RT and
PreAmp Kit■ SsoAdvanced
PreAmp Supermix■ Ordering Information

Pages 40–49■

Instrument Compatibility
PCR Tubes and Strips■ Hard-Shell PCR Plates■ PCR Plates■ PCR Plate Sealing■ Ordering Information■

PCR PLASTIC
CONSUMABLES■

7

Pages 50–59
High-Performance Assay Solutions
PrimePCR Real-Time PCR Products■ Assay Performance Standards and Validation■ Predesigned mRNA Pathway Panels■ PrimePCR Panels for a Broad Rangeof Pathways and Disease States■ Predesigned and Custom PrimePCRLong Noncoding RNA (lncRNA) Arrays■ PrimePCR Long Noncoding RNA Assays■ Experimental Controls and Reference
Gene Assays■ PrimePCR Data Analysis■■

PrimePCR ASSAYSAND PANELSPCR RUN ANDDATA ANALYSIS

8

Pages 60–61■

Overview of CFX Systems
and Software1CELL LYSIS
SingleShot Cell Lysis RT-qPCR Kits provide
a complete and fast solution for generationof lysates from cell cultures. These lysates
are optimized for downstream one- or two-step
quantitative PCR (qPCR) reactions, and do not
require an RNA purification step.
■

■

■

2

bio-rad.com

Amplification Reagents and Plastics

Ready-to-use cell lysate up to 100,000 cells
in 20 minutes
Simple protocol for automated, high-throughput reverse
transcription qPCR (RT-qPCR) experiments
Available in one- or two-step SYBR® Green or probes kits

CELL LYSIS

SingleShot Cell Lysis RT-qPCR Kits      

1SingleShot Cell Lysis RT-qPCR Kits
All SingleShot Cell Lysis RT-qPCR Kits feature:■ Complete removal of genomic DNA (gDNA) without
the need for purification
Preservation of RNA integrity by our potent blendof RNase inhibitors■

No loss of rare transcripts from column purification■

Optimal accuracy and high sensitivity of qPCR data■■

buffer containing proteinase K + DNase
2 Add lysis
Incubate 10 min at room temperature

Seed cells

Validated to be compatible with PrimePCR Assays
and Panels in preamplification workflows using
PrimePCR PreAmp Assays

24 hr growth

Tissue culture plate

4

3

Heat 10 min

Transfer

Tissue culture plate

5

Perform reverse transcription

Perform qPCR

PCR plate

6

Analyze data

104

Comparable results to those obtained whenusing purified RNA

RFU

■

1

103
102

SingleShot Kits are available in the following formats:■ SingleShot Cell Lysis Kits
■ SingleShot Cell Lysis Two-Step RT-qPCR Kits
■ SingleShot Cell Lysis One-Step RT-qPCR Kits

0

10

20

30

40

CyclesSingleShot workflow.

SingleShot Kit

Features

Cell Lysis Kits■■■

Cell Lysis Two-Step RT-qPCR Kits

■
■

■
■

Cell Lysis One-Step RT-qPCR Kits■■■

Product Options

Cell lysis for RT-qPCR
RT-qPCR reagents sold separately
No RNA purification needed

100 reactions, catalog #1725080
500 reactions, catalog #1725081

Cell lysis, reverse transcription, and qPCR
iScript Advanced cDNA Synthesis Kit
for RT-qPCR included
SsoAdvanced Universal Supermix included
No RNA purification needed

SYBR® Green, 100 reactions, catalog #1725085
Probes, 100 reactions, catalog #1725090

Cell lysis, reverse transcription, and qPCR
iTaq Universal One-Step Kit included
No RNA purification needed

SYBR® Green, 100 reactions, catalog #1725095
Probes, 100 reactions, catalog #1725070

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3

1CELL LYSIS

One-Step and Two-Step SYBR® Green or Probes Formats

One-Step and Two-Step SYBR® Green or Probes Formats
SingleShot Cell Lysis Kits■

SingleShot Cell Lysis Two-Step RT-qPCR Kits

Kits rapidly generate cell lysates that are optimized for RT-qPCR analysis
without RNA purification■

Kits yield high-performance RT-qPCR data directly from cell culture lysatesin less than 2 hours after cell lysis■

Superior gDNA removal while preserving RNA integrity■

Available with SYBR® Green or probe chemistry■

Minimal hands-on protocol provides a high-throughput solution■

SingleShot RNA Control is included to ensure optimal input cells and lysates

Reproducibility of Cell Lysates

PCR Efficiency across Expression Levels
40

40Pearson r = 0.99

30

30
Cq

Replicate 2, Cq

35

25
20

20

10

15
0

10

20
Replicate 1, Cq

30

40

The SingleShot SYBR ® Green Kit demonstrates high reproducibility between technicallysate replicates. Two technical lysate replicates of four neuroblastoma cell lines (SH-EP, SK-N-AS,
NGP, and IMR-32) were examined using ten SYBR® Green qPCR Assays to evaluate the reproducibilityof the SingleShot SYBR® Green Kit. A Pearson correlation of 0.99 was observed, thus demonstrating
highly reproducible data between lysis reactions. Cq, quantification cycle.
Data used with permission from Gert Van Peer, Pieter Mestdagh, and Jo Vandesompele, Center
for Medical Genetics, Ghent University, Ghent, Belgium.

4

bio-rad.com

Amplification Reagents and Plastics

0

1

2

3

4

5

log input cells

SingleShot Probes Kit maintains high PCR efficiencies across all expression levels.
Accurate gene expression studies require that PCR efficiencies be within 90–110%, per
the minimum information for publication of quantitative real-time PCR experiments (MIQE)
guidelines. Using the SingleShot Probes Kit, three targets of varying expression levels wereanalyzed using input cell numbers ranging from 10 to 100,000 HeLa cells. Greater than 98%
PCR efficiencies were maintained regardless of the expression levels (TBP ▲, 99.6%;
HPRT ■, 98.6%; B2M ■, 99.2%) and sensitivity down to 10 cells was observed. The SingleShot
RNA Control (◆) was used to monitor PCR inhibition. As the control quantification cycle (Cq)values remained constant, no inhibition was noted.

CELL LYSIS

One-Step and Two-Step SYBR® Green or Probes Formats

1

One-Step and Two-Step SYBR® Green or Probes Formats■

Cell lysate up to 100,000 cells ready in 20 min■

Linear dynamic range across genes■

Available with SYBR ® Green or probe chemistry

35

30
PCR
R 2 Efficiency, %25SingleShot RNA Control is included to ensure
optimal input cells and lysates■ B2M 0.98
■ HPRT 0.98
■ TBP 0.98

20
1

2

3

4

98.2
99.1
98.9

5

log starting quantity

SingleShot SYBR ® Green One-Step Kit enables a large dynamic range.
Two technical lysate replicates were examined across three differentially expressed
genes (B2M, HPRT, TBP) using a human kidney tumor (HKT) cell line to evaluate thelinear dynamic range of the SingleShot SYBR® Green One-Step Kit. The kit showeda large dynamic range across all assayed expression levels. Cq, quantification cycle.
Dynamic Range Similar to Purified RNA

log RFU■

Dynamic Range across Expression Ranges

Cq

SingleShot Cell Lysis One-Step RT-qPCR Kits
■ Within 1.5 hr after cell lysis, yields high-performance
RT-qPCR data directly from cell culture lysates

102
0

10

20

30

40

CyclesVisit bio-rad.com/SingleShot or
download or request bulletins 6572
and 6604 for more information.

SingleShot SYBR® Green One-Step Kit lysates yield a dynamic range similar tothat of purified RNA. Gene expression data generated using SingleShot SYBR® Green
One-Step Kit (■) lysates and isolated RNA (Aurum Total RNA Mini Kit, ■) were comparedusing a human cell line (K652) and from 10 to 100,000 input cells. B2M expression wasassessed using a PrimePCR qPCR Assay. SingleShot SYBR® Green One-Step Kit lysates
showed a dynamic range similar to that of the RNA generated using the Aurum Total RNA
Mini Kit without the need for RNA isolation. RFU, relative fluorescence units.

Amplification Reagents and Plastics

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5

2 RNA ISOLATION
Bio-Rad provides two distinct technologies
for isolating total RNA. Silica membrane–based
kits, available in 96-well plate and spin column
formats, and PureZOL RNA Isolation Reagent,
which is ideal for scaling up isolation protocols.
■

■

■

6

bio-rad.com

Amplification Reagents and Plastics

 are designed and formulated to assist in the isolation
Kitsof highly pure and intact RNA from different starting materials
RNA is compatible with a variety of downstream applications– PCR or real-time PCR– Northern blotting– Microarray analysis– cDNA library construction
DNase treatment ensures gDNA removal

RNA ISOLATIONRNA Isolation Kits Selection Guide

2

RNA Isolation Kits Selection Guide
Aurum Total RNA Kits
Mini

Fatty and Fibrous Tissue

96

PureZOL RNA Isolation Reagent

Description

Guanidine isothiocyanate andb-mercaptoethanol efficiently lyse
samples and quickly inactivate
RNases

Kit combines PureZOL, which effectively
lyses tissues and cells, with the speedof silica membrane technology in a spincolumn format to yield high-quality,
intact RNA

Kit is composed of guanidine
isothiocyanate and b-mercaptoethanol
for efficient lysis and quick RNase
inactivation, followed by purification
in a 96-well plate

PureZOL efficiently lyses cells andtissues, deproteinates RNA, andinactivates endogenous nucleases
in a single step

FormatMini column
Filtration (vacuum or spin)

Mini column
Filtration (vacuum or spin)

96-well plate
Filtration (vacuum or spin)

Single solution organic extraction

Cultured cells

2 x 106

1 x 1071 x 106

1 x 107

Bacterial cells

2.4 x 109

2.4 x 109

8 x 108

2.4 x 109

Yeast cells

3 x 103 x 102 x 103 x 107

Hard animal tissue

20 mg

100 mg—

100 mgSoft to moderately hard animal tissue

40 mg

100 mg—

100 mgPlant tissue

Maximum starting material amounts

7

7

7

40 mg

100 mg—

100 mgIsolation method

Silica membrane

Lysis with PureZOL reagent, purification
on silica membrane

Silica membrane

Organic extraction

Number of preps

50 mini preps

50 mini preps

2 x 96-well plate

50 or 100 (1 ml/prep)

Number of washes

3

3

3

—

DNase I included*

Yes

Yes

Yes

No

DNase I digest time

15 min (animal tissue, 25 min)15 min10 min—Total preparation time**<50–80 min (with DNase I digest)<50–80 min (with DNase I digest)<60 min (with DNase I digest)<60 min

Binding capacity>100 µg>100 µg>40 µg—Elution volume

2 x 40 µl

2 x 40 µl80 µl30–100 µl* Removal not required.
** Total preparation time will vary depending on the tissue or cell type and which format is used (vacuum or spin).

For sample-specific yield information, visit
bio-rad.com/RNAselection and click
the RNA Isolation Selection Guide.

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7

2 RNA ISOLATION

Aurum Sample Preparation Kits and PureZOL RNA Isolation Reagent

Aurum Sample Preparation Kits and PureZOL RNA Isolation Reagent
Aurum Total RNA Kits

PureZOL RNA Isolation Reagent

Aurum Total RNA Kits are a family of isolation kits that provide a high yield of intact
RNA from a wide range of starting materials, including cultured cells, bacteria, andyeast, as well as plant and animal tissues.
■

■

■

■

PCR-ready RNA in less than 60 min
RNase-free reagents and plastic consumables ensure the integrityof isolated RNA■

Kit includes DNase I provided for removal of gDNA contamination■

Easy-to-use spin or vacuum protocol■

 ingle-solution format permits recovery of RNA from small quantities
Sof tissues or cells, making it ideally suited for gene expression studies
 fficient RNA purification from cultured cells, yeast, viruses, and bacteria,E
as well as plant and animal tissues
 ureZOL efficiently lyses cells and tissues, deproteinates RNA, and inactivatesP
endogenous nucleases in a single step■

Scalable starting sample amount■Convenient isolation of RNA, DNA, and protein from the same sample

Aurum Total RNA Isolation Products come in:■ Aurum Total RNA Fatty and Fibrous Tissue Kit■ Aurum Total RNA Mini Kit■ Aurum Total RNA 96 Kit

7326830, 50 preps
Aurum Total RNA Fatty and Fibrous Tissue Kit

For more information, download
bulletins 2919, 2920, and 5282.

8

bio-rad.com

Amplification Reagents and Plastics

7326820, 50 preps
Aurum Total RNA Mini Kit

7326800, 2 x 96-well preps
Aurum Total RNA 96 Kit

7326890, 100 ml
PureZOL RNA Isolation Reagent

RNA ISOLATION
Ordering Information

2

Ordering Information
Catalog #Description

73268307326870*73268207326800
73268807326890

Aurum Total RNA Fatty and Fibrous Tissue Kit
Aurum Total RNA Fatty and Fibrous Tissue Module
Aurum Total RNA Mini Kit
Aurum Total RNA 96 Kit
PureZOL RNA Isolation Reagent, 50 ml
PureZOL RNA Isolation Reagent, 100 ml* Not provided with PureZOL RNA Isolation Reagent (see catalog #7326880 or #7326890 to order separately).

Amplification Reagents and Plastics

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93 REVERSE TRANSCRIPTION
Bio-Rad reverse transcription kits provide
easy setup and high reproducibility forfirst-strand cDNA synthesis. Bio-Rad reverse
transcription reagents cover a rangeof priming strategies, including primer blends.
■

■

■

■

10

bio-rad.com

Amplification Reagents and Plastics


Formulated
for efficient reverse transcription acrossa broad linear dynamic rangeP otent RNase A inhibitors protect RNA during setup
and reverse transcriptionF lexible input RNA capacity to suit different
experimental needs ptimized for gene expression analysis usingO
real-time qPCRREVERSE TRANSCRIPTION
Reverse Transcription Kit Selector

3

Reverse Transcription Kit Selector

Features

Applications

■
■

■
■

Reliance Select cDNA
Synthesis Kit

iScript Reverse
Transcription Supermix
for RT-qPCR

iScript gDNA Clear
cDNA Synthesis Kit

iScript Advanced cDNA
Synthesis Kit for
RT-qPCR

iScript SelectcDNA Synthesis Kit

iScript cDNA
Synthesis Kit

Overcome challenging
samples

Minimum reaction setup
time

Effective gDNA removal
before reverse transcription

Maximum input RNA
for high cDNA yields

Flexible priming options
and high fidelity

Reliable value solution

CloningSequencing and nextgeneration sequencing
Gene expression
RNA quantification■■

Gene expression
RNA quantification■■

Gene expression
RNA quantification■■

Gene expression
RNA quantification

Cloning
Sequencing andnext-generation
sequencing■ Gene expression■ RNA quantification
■

■

■

■

Gene expression
RNA quantification

Total Input
RNA Range

2 μg–100 fg

1 µg–1 pg

1 µg–1 pg

7.5 µg–100 fg

1 µg–1 pg

1 µg–100 fg

Kit Format

6 tubes

1 tube3 tubes

2 tubes

5 tubes

2 tubes

Blend of random primers
and oligo(dT)

Blend of random primers andoligo(dT)Blend of random primers
and oligo(dT)Priming
Options■■■

Random primers
Oligo(dT)Gene specificOptimalReaction
Temperature

50°C

DNase Step
Included

✔

■
■
■

46°C

46°C

Random primers
Oligo(dT)Gene specific

Blend of random primers
and oligo(dT)

46°C

42°C

46°C

✔

Time toProduce cDNA

10 min26 min36 min21 min15–65 min26 minRNase H+
Activity

Reduced

✔

✔

✔

✔

✔

Go to bio-rad.com/PCRReagentSelector
and use our interactive selection tool to findthe right reverse transcriptase for your needs.

Amplification Reagents and Plastics

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11

3 REVERSE TRANSCRIPTION
Reliance Select cDNA Synthesis Kit

Reliance Select cDNA Synthesis Kit
product featuring Reliance Reverse Transcriptase, a novel
chimeric enzyme that delivers superior results across a broad
dynamic range, even with challenging samples.
■

■

■

12

 reate cDNA libraries from a wide range of inputs and a varietyCof sample types
Overcome reaction inhibitors
 ower through complex secondary structures and GC-richPregions (up to 80%)■

Eliminate gDNA for more accurate RT-qPCR■

Shorten time to results■

Optimize experimental design with flexible priming strategies

bio-rad.com

Amplification Reagents and Plastics

Unrivaled Performance in the Presence of Inhibitors
4.00
DCq = Cq(treated) – Cq(untreated)

The Reliance Select cDNA Synthesis Kit is a high-performance

3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Heparin,0.005 U/ml

Formalin,
0.125%

Ethanol,
7.5%

TRIzol,0.3%

Guanidinium Ammonium
isothiocyanate, acetate,0.05 M
0.2 M

The Reliance Select cDNA Synthesis Kit demonstrates superior efficiency compared to the SuperScript IV
First-Strand Synthesis System (Thermo Fisher Scientific Inc.) in the presence of inhibitors. The Reliance SelectKit ( ■ ) delivers more consistent results and earlier average Cq values than the SuperScript IV System ( ■ ). This isevident from the lower ΔCq values of treated versus untreated reactions.

REVERSE TRANSCRIPTION

Reliance Select cDNA Synthesis Kit

3

Reliance Select cDNA Synthesis Kit
Unbiased Reverse Transcription and Superior Sensitivity

Greater Reverse Transcription Efficiency with Degraded Samples
DCq = Cq(degraded) – Cq(intact)

RFU

104

103

1

2

3

3	SuperScript IV System
No background RNA
4	1 μg background RNA

20

30

1.50
1.00
0.50
0.00
ACT

GAPDH

TUB

The Reliance Select cDNA Synthesis Kit delivers earlier Cq values compared to the SuperScript IV
System when tested with artificially degraded RNA samples. The Reliance Select Kit ( ■ ) delivers moreaccurate results between degraded and intact RNAs than the SuperScript IV System ( ■ ). This is evident fromthe lower ΔCq values of degraded versus intact reactions.

2 1 μg background RNA

102

2.50
2.00

18S

1	Reliance Select Kit
No background RNA

4

3.50
3.00

40

CyclesThe Reliance Select cDNA Synthesis Kit allows for sensitive detection of low-abundance
transcripts. The Reliance Select Kit allows for more sensitive detection of External RNA Controls
Consortium (ERCC) spike-in RNAs in the presence of background RNA compared to theSuperScript IV System. RFU, relative fluorescence units.

Ordering Information
Catalog #Description

12012802 Reliance Select cDNA Synthesis Kit, 25 x 20 μl reactions
12012801 Reliance Select cDNA Synthesis Kit, 100 x 20 μl reactions
17006111 Reliance Select cDNA Synthesis Kit, 500 x 20 μl reactions

For more information, download
bulletin 7330.

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3 REVERSE TRANSCRIPTION
iScript cDNA Synthesis Kits

iScript cDNA Synthesis Kits
iScript PerformanceSuperior 5' to 3' Coverage

iScript Reverse Transcription Kits were designed for easy setup, highreproducibility, and bias-free cDNA synthesis. iScript reagent features
include:■■

RNase H+ activity of the reverse transcriptase ensures 1:1 RNA:cDNA 		
conversion, resulting in greater sensitivity and more accurate data

103
RFU

■

Optimal blend of oligo(dT) and random primers for complete
and unbiased coverage

102

Potent blend of RNase inhibitors prevents RNA degradation
during reaction setup and cDNA synthesis steps

0

5

10

15

20
Cycles25

30

35

Total RNA (7.5, 2, 1, and 0.1 µg) from HeLa cells was reverse transcribed using the iScript Advanced cDNA
Synthesis Kit for RT-qPCR in a 20 µl reaction. One-tenth of the generated cDNA was used as template to amplify
the APC gene in a 10 µl qPCR reaction with iTaq Universal SYBR® Green Supermix on a CFX384 Real-Time PCR
System. Primer pairs were designed at the 5' end ( ■, 91 bp amplicon) and 3' end ( ■, 69 bp amplicon). There was no
significant difference between the two primer pairs. This demonstrates superior coverage of the 5' and 3' regions oftarget mRNA. RFU, relative fluorescence units.
Broad Dynamic Range
104

RFU

— 1 µg— 100 ng
— 10 ng— 1 ng— 100 pg— 10 pg— 1 pg103
R 2 = 0.999
Efficiency = 99.7%
Slope = –3.33

For more information, download
bulletins 6031, 6125, and 6742.

Watch the tutorial!
Understanding Reverse Transcription
bio-rad.com/RT-tutorial

14

bio-rad.com

Amplification Reagents and Plastics

0

10

20

30

40

CyclesiScript Reverse Transcription Supermix for RT-qPCR efficiently reverse transcribes RNA over a broad linear
dynamic range for reliable gene expression analysis data. Different amounts of HeLa cell RNA (amounts shownin inset) were reverse transcribed and one-tenth of the resulting cDNA was used as a template to amplify the b-actin
gene (~90 bp) in 20 µl qPCR reactions with iTaq Universal SYBR® Green Supermix. RFU, relative fluorescence units.

REVERSE TRANSCRIPTION
iScript cDNA Synthesis Kits

3

iScript cDNA Synthesis Kits
iScript Reproducibility

104

Effective Removal of Genomic DNA

RNAAverage Cq

SD

CV, %

6,000

100 ng100 pg

21.35
31.56

0.123
0.147

0.576
0.465

5,000
+RT

10

10

2

RFU

RFU

4,000
3

3,000
2,000
1,000

10

20

30

40

–RT

0
0

10

CyclesExcellent data reproducibility. PGK-1 mRNA (~160 bp), a gene that encodes a glycolytic enzyme,
was quantified using iScript Reverse Transcription Supermix for RT-qPCR with both 100 ng ( ■ )and 100 pg ( ■ ) of input RNA. For each input RNA, 48 individual RT reactions were performed andone-tenth of the resulting cDNA was used in the qPCR reaction with SsoFast Probes Supermix.
The gene expression analysis data show excellent reproducibility with both high and low levels of
input target mRNA. The ~10 quantification cycle (Cq) difference for the 1,000-fold dilution of RNA(100 ng–100 pg) demonstrates good reverse transcription efficiencies across different input RNAs.
CV, coefficient of variation; RFU, relative fluorescence units; SD, standard deviation.

20
Cycles30

40

iScript gDNA Clear cDNA Synthesis Kit eliminates contaminating genomic DNA.
HeLa RNA (1 µg) was spiked in with 300 ng gDNA. Contaminating gDNA was cleared and cDNA
synthesis was then performed with (+RT) and without (–RT) reverse transcriptase using iScript
gDNA Clear cDNA Synthesis Kit. qPCR was performed using the human TUBA assay, an exonic
assay that detects both mRNA and gDNA. Complete removal of gDNA is demonstrated by theabsence of amplification in the –RT sample. RFU, relative fluorescence units.

Ordering Information
Catalog #Description
cDNA Synthesis Reagents
1725037
iScript Advanced cDNA Synthesis Kit for RT-qPCR, 25 x 20 μl reactions
1725038
iScript Advanced cDNA Synthesis Kit for RT-qPCR, 100 x 20 μl reactions
1725038BUN iScript Advanced cDNA Synthesis Kit for RT-qPCR, 500 x 20 µl reactions
1708890
iScript cDNA Synthesis Kit, 25 x 20 μl reactions
1708891
iScript cDNA Synthesis Kit, 100 x 20 μl reactions
1725034
iScript gDNA Clear cDNA Synthesis Kit, 25 x 20 µl reactions
1725035

iScript gDNA Clear cDNA Synthesis Kit, 100 x 20 μl reactions

1725035BUN

iScript gDNA Clear cDNA Synthesis Kit, 500 x 20 µl reactions

Catalog #Description

1708840

iScript Reverse Transcription Supermix for RT-qPCR, 25 x 20 μl reactions

1708841

iScript Reverse Transcription Supermix for RT-qPCR, 100 x 20 μl reactions

1708841BUN

iScript Reverse Transcription Supermix for RT-qPCR, 500 x 20 µl reactions

1708896

iScript Select cDNA Synthesis Kit, 25 x 20 μl reactions

1708897

iScript Select cDNA Synthesis Kit, 100 x 20 μl reactions

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4 PCR AND REAL-TIMEPCR REAGENTS

Bio-Rad PCR and real-time PCR reagents and kits
feature advanced enzyme technology andbuffer formulation for robust amplification anddetection with maximum sensitivity, specificity,and efficiency.
■

■

■

■

■

16

bio-rad.com

Amplification Reagents and PlasticsA ntibody-mediated hot-start technology enables instantpolymerase activation and superior specificity
Patented Sso7d fusion enzyme technology delivers
higher processivity and inhibitor toleranceFaster time to results without compromising qPCR data quality
 igh performance obtained on any instrument, under any reactionH
and/or cycling conditions
A novel chimeric Reliance Reverse Transcriptase delivers superior
one-step RT-qPCR results, even with challenging samples

PCR AND REAL-TIME PCR REAGENTS
Real-Time PCR Reagents Selection Guide

4

Real-Time PCR Reagents Selection Guide
Use this selection guide to choose an appropriate reagent for your application.
qPCR (DNA to Cq)
SsoAdvanced Universal
Inhibitor-Tolerant Supermix

SsoAdvanced
Universal Supermixes
Robust Sso7d-driven performancewith demanding templates andunder challenging conditions

Features

One-Step RT-qPCR (RNA to Cq)

Sso7d-driven optimal
performance for samples
containing PCR inhibitors

Reliance One-Step
Multiplex Supermix

iTaq Universal Supermixes
Advanced formulation for reliable,
reproducible performance

Single-tube, 4x supermix for
5-plex reaction with 24 hr
assembled reaction stability

iTaq Universal One-Step Kits
Easy 2-tube system for SYBR®
Green– and probe-based onestep RT-qPCR

Sensitive detection of
low-abundance targets

•••••

•••••

•••••

•••••

•••••

Efficient amplification ofdifficult targets

•••••

•••••

•••

•••••

•••

Specific amplification oftargets

•••••

•••••

•••••

•••••

•••••

Robust performance undervarying PCR conditions*

•••••

•••••

•••

•••

•••

Tolerance to PCR inhibitors

••••

•••••

•••

••••

•••

Fast cycling compatibility

Yes

Yes

Yes

Yes

Yes

Available detection
chemistrySYBR® Green and probes

SYBR® GreenSYBR® Green and probes

ProbesSYBR® Green and probes

Compatibility with any
qPCR instrument

Any instrument

Any instrument

Any instrument

Any instrument

Any instrument■■■

Samples■■••• Fair•••• Good

Blood spot
Cell lysate
Crude
extraction
FFPE
LCM

■
■
■
■

Plant extract
Plant tissue
Plasmid
Purified
gDNA and
cDNA

■
■
■

■
■

Blood spot
Cell lysate
Crude
extraction
FFPE
LCM

■
■
■
■

Plant extract
Plant tissue
Plasmid
Purified
gDNA andcDNA■■

Plasmid
Purified gDNA and cDNA■■

Cell lysate up to 10 µl
Purified RNA■■

Cell lysate up to 4 μl
Purified RNA••••• Excellent

cDNA, complementary DNA; Cq, quantification cycle; FFPE, formalin-fixed paraffin-embedded; gDNA, genomic DNA; LCM, laser capture microdissection; RT-qPCR, reverse transcription quantitative PCR.
* Primer concentrations, annealing temperatures, salt concentrations, and/or cycling protocol (standard or fast).

Go to bio-rad.com/PCRReagentSelector
and use our interactive selection tool to findthe right product for your needs.

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17

4 PCR AND REAL-TIME PCR REAGENTS
SsoAdvanced Universal Supermixes

SsoAdvanced Universal Supermixes
Innovative Sso7d Fusion Enzyme Technology for Robust qPCR■

SsoAdvanced Universal Supermixes are exclusive high-performance real-time
PCR reagents based on the Bio-Rad patented* Sso7d fusion protein polymerase
technology and advanced buffer formulation.
The supermixes are uniquely formulated to provide higher processivity, increased
PCR inhibitor tolerance, and robust performance with challenging templates andtargets in SYBR® Green– or probe-based qPCR. They deliver superior performancefor a wide range of applications on any qPCR platform and are validated to ensure
optimal results with Bio-Rad PrimePCR Gene Expression Assays.

Use any real-time PCR system — the universal reference dye inthese supermixes enables ROX normalization of qPCR data
regardless of the ROX requirements of the qPCR system

SsoAdvanced Universal Supermixes include:■ SsoAdvanced Universal SYBR® Green Supermix■ SsoAdvanced Universal Probes Supermix■ SsoAdvanced Universal Inhibitor-Tolerant SYBR® Green Supermix

Polymerase

SsoAdvanced Universal Supermixes allow you to:■ Achieve superior real-time PCR results under various experimental
conditions — our robust formulation delivers consistent performancein fast and standard cycling across a broad range of reaction conditions, primer
concentrations, and temperature ranges■■■

I ncrease qPCR sensitivity and efficiency of detection fromcompromised samples — the highly processive Sso7d fusion polymerase
exhibits increased resistance to a wide variety of PCR inhibitors, enhancing
sensitivity and overall performance
Shorten time to results while maintaining qPCR data quality — the Sso7d
fusion polymerase and optimized buffer provide rapid polymerization kinetics
and instant polymerase activation

The dsDNA binding protein, Sso7d, stabilizes the polymerase-template complex,
increases processivity, and provides greater speed and reduced reaction times
compared to conventional DNA polymerases. Sso7d fusion polymerases are significantly
more resistant to PCR inhibitors, making the SsoAdvanced Supermixes ideal choices forchallenging applications, such as direct qPCR, without the need for sample preparation.

Carry out high-performance singleplex and duplex reactions — the Sso7d 		
fusion polymerase and advanced formulation enable robust performancein singleplex or duplex real-time PCR reactions, providing the highest data 		
13-0478 Sso7d Illustration Final
precision and allowing cost and time savings* U.S. patents 6,627,424; 7,541,170; and 7,560,260.
02.20.13

Watch the tutorial! Understanding
Real-Time PCR Supermixes
bio-rad.com/RTsmxTutorial

18

Sso7d

bio-rad.com

Amplification Reagents and Plastics

Visit bio-rad.com/Supermixes or
download or request bulletin 7178 formore information.

PCR AND REAL-TIME PCR REAGENTS
SsoAdvanced Universal Supermixes

4

SsoAdvanced Universal Supermixes
SsoAdvanced Universal SYBR® Green and Probes Supermixes
SsoAdvanced Universal Supermixes employing our patented* Sso7d fusion enzyme technology provide higher processivity, increased PCR inhibitor
tolerance, and robust performance with challenging templates and target sequences in real-time PCR.
SsoAdvanced Universal SYBR® Green Supermix

SsoAdvanced Universal Probes Supermix

A. Optimal PCR Sensitivity

A. Superior Multiplex qPCR Performance
10

104

ΔRn

RFU

1
103

0.1

102

18S rRNA efficiency = 98%
TFRC efficiency = 101%
0

0.01

40

30

20

10

0

10

Cycles

20

30

40

30

40

CyclesB. Single-Copy Detection

B. Superior PCR Specificity

104

2,000

RFU

– d (RFU) /dT

1,500
1,000

103

500
102
0

65

70

75

80
Temperature, °C

85

90

95

Optimal PCR sensitivity and specificity with SsoAdvanced Universal SYBR® Green
Supermix. A, a PrimePCR Assay targeting b2 microglobulin, using 250, 125, 62.5, 31.25,
15.625, and 7.8125 cells/20 µl reaction, was performed on a CFX96 Real-Time PCR
System and yielded optimal PCR sensitivity; B, melt curve analysis displays enhanced
PCR. RFU, relative fluorescence units.

0

10

20
CyclesSuperior qPCR performance in a duplex reaction (A) and single-copy detection (B)with SsoAdvanced Universal Probes Supermix. A, predesigned TaqMan Gene Expression
Assays in a duplex reaction with 10 ng–100 fg cDNA using an 18S rRNA assay (■) and
100 ng–10 pg cDNA using a TFRC assay (■) were performed on an Applied Biosystems
ViiA 7 System; B, a gDNA assay targeting IL-1b, with gDNA serially diluted to a calculated
single copy (■), was performed on a CFX96 Real-Time PCR System. RFU, relative
fluorescence units; ΔRn, baseline-corrected normalized reporter.

* U.S. patents 6,627,424; 7,541,170; and 7,560,260.

Amplification Reagents and Plastics

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19

4 PCR AND REAL-TIME PCR REAGENTS
SsoAdvanced Universal Supermixes

SsoAdvanced Universal Supermixes
SsoAdvanced Universal Inhibitor-Tolerant SYBR® Green Supermix
SsoAdvanced Universal Inhibitor-Tolerant SYBR® Green Supermix is a highperformance real-time PCR supermix specifically formulated for use with difficult
target sequences in a wide range of challenging samples, including crude lysates.
■

■

■

20

Power through PCR inhibitors — delivers unrivaled performance with common
PCR inhibitors, such as heparin, polysaccharides, culture media, and more
Skip RNA and DNA extraction by using crude samples — lysates from plants,
seeds, cells, bacteria, or FFPE samples
Increase cDNA loading — add up to 20% cDNA from our iScript Advanced cDNA 		
Synthesis Kit for RT-qPCR or iScript Reverse Transcription Supermix for RT-qPCR

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Amplification Reagents and Plastics

Applications and Uses
Environmental monitoring
Gene expression■ Genetically modified organism (GMO) testing■ Mutation detection■ Pathway analysis■ Single nucleotide polymorphism (SNP) genotyping■ Viral and bacterial detection■■

PCR AND REAL-TIME PCR REAGENTS
SsoAdvanced Universal Supermixes

4

SsoAdvanced Universal Supermixes
SsoAdvanced
Universal InhibitorTolerant SYBR®
Green Supermix

Quanta Bioscience
PerfeCTa SYBR®
Green FastMix

SsoAdvanced
Universal InhibitorTolerant SYBR®
Green Supermix

Clontech TerraqPCR Direct SYBR®
PremixQuanta Bioscience
PerfeCTa SYBR®
Green FastMix

Clontech TerraqPCR Direct SYBR®
PremixSampleAssay

Challenging Target Assay (hsGAPDH [226 bp]; 5 ng hgDNA)

SampleAssay

Challenging Target Assay (hsGAPDH [226 bp]; 5 ng hgDNA)

Bile salts

Cholic acid

0.5 μg/μl

0.25 μg/μl

0.5 μg/μl

Cell lysates

MEM+FBS

5%

5%

5%

0.25 μg/μl

0.125 μg/μl

0.25 μg/μl

PBS

10%

10%

5%

0.3125 μg/μl

Trypsin

0.000094%

0.000094%

0.00%

NaAc

25 mM

25 mM

12.5 mM

Algae

Deoxycholic acid
Polygalacturonicacid
Cholesterolhydrochloride
Alginic acidNaCl

12.5 mM

6.25 mM

6.25 mM

Clay

EtOH

2.50%

1.25%

1.25%

Blood

Isopropanol

2.50%

1.25%

1.25%

TRIzol

0.30%

0.30%

0.15%

InstaGene Matrix

0.30%

0.15%

0.02%

Green tea

0.50%

—

—

Chocolate

0.13%

—

—

Dust

0.18 μg/μl

—

—

SDS

0.003%

0.003%

0.006%

DMSO

3%

1.50%

1.50%

DTT

2 mM

2 mM

No amplification

Spermidine

1.25 mM

1.25 mM

1.25 mM

Urea

80 mM

80 mM

80 mM

Polysaccharides
Lipids

0.625 μg/μl

0.1563 μg/μl

0.125 μg/μl

0.0625 μg/μl

0.01563 μg/μl

0.25 μg/μl

0.125 μg/μl

0.25 μg/μl

Montmorillonite

20 ng/μl

20 ng/μl

20 ng/μl

Heparin

5 ng/μl

0 ng/μl

2.5 ng/μl

Hematin1.5625 μM

0.7813 μM

0.7813 μM

EDTA

0.7813 mM

0.7813 mM

0.3907 mM

Serum

0.63%

—

—

Soil

Humic acid

0.625 ng/μl

0.1563 ng/μl

0.3125 ng/μl

TextileIndigo5 mM

5 mM

2.5 mM

Wine

Tannic acid

0.6250 ng/μl

0.3125 ng/μl

0.3125 ng/μl

PlantsCellulose
Pectin (for fiber
control)

2%

1%

1%

0.125 μg/μl

0.125 μg/μl

0.25 μg/μl

Hair, tissues

Melanin

20 ng/μl

10 ng/μl

10 ng/μl

Bones, teeth

CaCl2

1.25 mM

0.3125 mM

0.6250 mM

Samplepreparation

Miscellaneous

Body fluids

PCR inhibitor tolerance testing with SsoAdvanced Universal Inhibitor-Tolerant SYBR® Green Supermix against Quanta Bioscience PerfeCTa SYBR® Green FastMix and Clontech Terra qPCR Direct SYBR® Premix. Each qPCR
reaction was performed using a challenging long (226 bp) human gDNA assay targeting GAPDH and 5 ng of human gDNA. The values represent the maximum percentage and concentration for which a ≤1 quantification cycle (Cq) delay was
noted. Any delay >1 Cq was deemed unacceptable for inhibitor tolerance. —, not tested.

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21

4 PCR AND REAL-TIME PCR REAGENTS
iTaq Universal Supermixes

iTaq Universal Supermixes
iTaq Universal Supermixes are ready-to-use, 2x reaction master mixes
for SYBR® Green– or probe-based qPCR. These supermixes contain anadvanced buffer formulation for optimal qPCR performance.

Matchless Precision and Accuracy
A

■

■

■

Obtain reliable and reproducible gene expression analysis results
Achieveconsistent results across real-time instrument platforms
under fast or standard conditions

Employ PrimePCR Assays and Panels — validated for use with
iTaq Universal Supermixes

■

■

22

10

0

 tilize any real-time PCR system, with its blend of passiveUreference dyes

iTaq Universal SYBR® Green Supermix
■

103

102
20
Cycles

30

40

25

30

B

RFU

■

 uickly start your experiment within 30 sec with antibody-mediatedQ
hot-start iTaq DNA Polymerase for rapid activation

RFU

104
■

103

Contains an advanced buffer formulation with SYBR® Green I for optimal
qPCR performance
Provides a way to amplify gDNA and difficult amplicons with superior efficiency
Applications and uses include gene expression analysis, genotyping,detection, and pathogen detection

bio-rad.com

Amplification Reagents and Plastics

15

20
CyclesSuperior accuracy and precision with iTaq Universal SYBR® Green Supermix ensures productionof high-quality data. An ACTB cDNA assay was performed on a CFX96 Real-Time PCR Systemusing iTaq Universal SYBR® Green Supermix, which produced 1.33-fold discrimination. RFU, relative
fluorescence units.

PCR AND REAL-TIME PCR REAGENTS
iTaq Universal Supermixes

4

iTaq Universal Supermixes
iTaq Universal Probes Supermix■■■

 arry out high-performance singleplex and duplex reactions — robustC
performance in singleplex or duplex real-time PCR reactions enables increased
data precision and saves samples, reagents, and time chieve superior results under various conditions — broad range of cyclingA
conditions, primer concentrations, temperature ranges, sample types, andtarget sequences can be applied ecrease run times and time to results without compromising qPCR
D
data quality — provides rapid polymerization kinetics and instant polymerase
activation for run times less than 40 minutes

Maximum Dynamic Range for Complete Gene Expression Detection

Advantageous Sensitivity Compared to TaqMan Master Mix

1
ΔRn

ΔRn

1

0.1

R 2 = 0.999
Efficiency = 99.4%
0

10

20

30

40

0.1

0

10

20

30

40

CyclesCyclesDynamic range of iTaq Universal Probes Supermix. An ACTB cDNA assay was performedon an Applied Biosystems 7900 System and produced standard curve R 2 = 0.999 andefficiency = 99.4% with a 6-log dynamic range. ∆Rn, baseline-corrected normalized reporter.

Comparison of dynamic range using iTaq Universal Probes Supermix vs. TaqMan
Fast Universal PCR Master Mix (Thermo Fisher Scientific). An ACTB cDNA assay was
performed on an Applied Biosystems 7900 System. iTaq Universal Probes Supermix ( ■)demonstrates a 6-log dynamic range with earlier Cq values and greater sensitivity
compared to TaqMan Fast Universal PCR Master Mix ( ■), which demonstrates a 5-log
dynamic range and delayed Cq values. ∆Rn, baseline-corrected normalized reporter.

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23

4 PCR AND REAL-TIME PCR REAGENTS
Reliance One-Step Multiplex RT-qPCR Supermix

Reliance One-Step Multiplex RT-qPCR Supermix
Reliance One-Step Multiplex Supermix is a ready-to-use, 4x reaction mix
optimized for sensitive amplification of up to five targets in a single reaction.
Expertly formulated with an optimal enzyme blend, including Reliance
Reverse Transcriptase, Sso7d DNA polymerase, and an advanced reaction
buffer, this robust supermix delivers reproducible RT-qPCR performancein a multiplex fashion even with challenging templates, difficult targets, orin the presence of PCR inhibitors. With its exceptional room temperaturestability for assembled reactions, it can be easily integrated into an
automated workflow. Reliance One-Step Multiplex Supermix is an ideal
solution for multiplexed RT-qPCR, especially for pathogen/viral detection
and high-throughput screening applications.
Key features and benefits of this robust supermix include:
■

■

■

■

■

24

 ensitive, reproducible multiplexing of up to 5 targets — gets theS
most out of your samples while reducing costs oom temperature stability of assembled reaction up to 24 hours —Renables use in an automated workflow to maximize throughput
 onvenient 1-tube supermix — minimizes reaction setup time andChandling errors x concentrated formulation — increases detection sensitivity by
4
maximizing input volume
 olerant to common PCR inhibitors — for use with lysates or samplesTof varying quality

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Amplification Reagents and Plastics

Reliance One-Step Multiplex Supermix allows researchers to:■

Amplify multiple targets with maximum efficiency■

Accelerate data throughput while decreasing costs and reaction time■Achieve consistent and reproducible data, run after run■

Streamline automated workflows by bypassing the need for plate refrigeration

PCR AND REAL-TIME PCR REAGENTS
Reliance One-Step Multiplex RT-qPCR Supermix

4

Reliance One-Step Multiplex RT-qPCR Supermix
Exceptional Performance of Five-Target Multiplex

Reliable and Sensitive Ten-Copy Detection

40
104

35

RFU

Cq

30
25

103
20
15
10

102
–3

–2

–1
0
1
log starting quantity, ng

2

3

0

10

20
Cycles30

40

Reporter Dye

Assay

Efficiency, %

R2

Slope

Reporter Dye

Assay

Template

Input Amount(s)

— FAM

ACTB

99.0

1.000

–3.346— FAM

Control

Synthetic RNA

10 copies/reaction

— HEX

NGFRP

100.5

0.999

–3.311— FAM

Control

Synthetic RNA— Texas Red

TBP

97.6

0.997

–3.382

— HEX

NGFRP

HeLa RNA

— Cy5

EF1a

96.6

0.999

–3.406— Texas Red

HSPA4

HeLa RNA— Cy5.5

GAPDH

96.9

1.000

–3.397

— Cy5

EF1a

HeLa RNA— Cy5.5

GAPDH

HeLa RNA

Fig. 1. The linear dynamic range in 5-plex one-step RT-qPCR reactions.
Five targets across 7 orders of magnitude (1 pg–1 μg input RNA) were amplified using
Reliance One-Step Multiplex Supermix on a CFX96 Touch Real-Time PCR System. The
results demonstrated exceptional performance with superior efficiency and linearity
over a wide dynamic range down to 1 pg input RNA. Cq, quantification cycle.

Four 13.5-fold serial
dilutions from 1 μg

Fig. 2. Detection of ten copies of synthetic RNA quantified by one-step reverse
transcription Droplet Digital PCR (RT-ddPCR). Fifty-three replicates of ten copies
of synthetic RNA (red circle) along with 5-plex reactions consisting of one target fromsynthetic RNA and four targets from HeLa RNA across the above serial dilutions were
amplified using Reliance One-Step Multiplex Supermix. The results demonstrated
reliable and sensitive detection of RNA targets down to 10 copies/reaction with tightCq values between the replicates. RFU, relative fluorescence units.

For more information, download
bulletin 7175.

Amplification Reagents and Plastics

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25

4 PCR AND REAL-TIME PCR REAGENTS
iTaq Universal One-Step Kits

iTaq Universal One-Step Kits

■

■

■

Achieve superior gene expression results under various cycling
conditions — our robust formulation delivers consistent performance instandard or fast cycling conditions, primer concentrations, sample and target
types, and temperature ranges, allowing cost and time savings
Improve PCR efficiency and obtain wider dynamic range, superior sensitivity
and specificity, and inhibitor tolerance — even with cell lysates — without
affecting performance se any ROX dependent or independent real-time PCR system — the universal
Ureference dye in these supermixes enables ROX normalization of qPCR data
regardless of the ROX requirements of the qPCR system
Minimize handling and contamination risk — perform cDNA synthesisand qPCR in 1 tube
iTaq Universal SYBR® Green One-Step Kit■■

Benefit from enhanced efficiency, specificity, and sensitivity — our 		patented* inhibitor reducer prevents the interference of the RT enzyme 		with the DNA polymerase during the replication of cDNA btain better results with qPCR assays — simplify your workflow andOexperimental design using our wet-lab validated PrimePCR Assays; eachassay for the human, mouse, and rat genomes was experimentally tested
for optimal efficiency, specificity, sensitivity, and linear dynamic range* U. S. patent 8,338,094.

26

bio-rad.com

Amplification Reagents and Plastics

A. Bio-Rad
10

ΔRn

■

Superior Dynamic Range and Sensitivity

1

R2 = 0.997
Efficiency = 103%

0.1
0

10

20
Cycles30

40

B. Thermo Fisher Scientific
10

ΔRn

iTaq Universal One-Step Kits are a fast and convenient solution for real-time
RT-qPCR, using the powerful combination of iScript RNase H+ Moloney Murine
Leukemia Virus (MMLV) Reverse Transcriptase and hot-start iTaq DNA Polymerase
in one reaction. Select from SYBR® Green– or probe-based assays for flexibility.

1

R2 = 0.922
Efficiency = 159%

0.1
0

10

20
Cycles30

40

Superior dynamic range and sensitivity with iTaq Universal SYBR® Green One-Step Kit. A 120 bp
human FAS assay with an input of 500 ng–50 pg total RNA was performed on a ViiA 7 Real-Time PCR
System (Thermo Fisher Scientific). A, iTaq Universal SYBR ® Green One-Step Kit; B, Power SYBR® Green
RNA-to-CT 1-Step Kit (Thermo Fisher Scientific). Note the delayed threshold cycle (CT ) values, loss of dynamic
range, and compression of input 500 ng total RNA using the Power SYBR ® Green RNA-to-CT 1-Step Kit.
This demonstrates RT-qPCR inhibition with higher input RNA and lack of low-level target expression detection.
ΔRn, baseline-corrected normalized reporter.

PCR AND REAL-TIME PCR REAGENTS
iTaq Universal One-Step Kits

4

iTaq Universal One-Step Kits
iTaq Universal Probes One-Step Kit■■

Obtain superior results with singleplex and multiplex reactions — advanced formulation enables
the simultaneous amplification of up to 3 targets, resulting in higher data precision with fewer pipetting
steps and reduced sample usage
 igh-throughput real-time PCR screening and validation — simplified workflow and reduced cyclingH
times enable screening and validation of the greatest number of samples and targets in the shortest
period of time

Optimal RT- qPCR Efficiency and Sensitivity with an AT-Rich and Long Amplicon

Superior RT-qPCR Performance in a Triplex Reaction

A. Bio-Rad

A. Bio-Rad
Dye

103

RFU

RFU

104

R2 = 0.999
Efficiency = 94.1%

102
0

10

20

■ FAM
■ HEX
■ Quasar

103

0

10

20

logs

Efficiency, %Standard Curve, R 2

4
4
4

96.7
96.7
95.6

0.999
0.999
0.999

30

Dye

104

RFU

RFU

0.998
0.999
0.998

B. Thermo Fisher Scientific4103

R2 = 0.996
Efficiency = 93.3%

102
10Standard Curve, R 2

106
101
103

CyclesB. Thermo Fisher Scientific0

Efficiency, %

5
4
6

102

30

Cycles

10

logs

20
Cycles30

Optimal RT-qPCR efficiency and sensitivity with iTaq Universal
Probes One-Step Kit. A 61% AT-rich, long, 248 bp HPRT assay withan input of 100 ng–10 pg RNA was performed on a CFX96 Real-Time
PCR System. A, iTaq Universal Probes One-Step Kit; B, TaqMan RNA-to-CT
1-Step Kit (Thermo Fisher Scientific) produced delayed Cq values with lessthan optimal data precision for the technical replicates. RFU, relative
fluorescence units.

■ FAM
■ HEX
■ Quasar

103

102
0

10

20
Cycles30

Superior RT-qPCR performance in a triplex reaction with iTaq Universal Probes One-Step Kit. An XPO (FAM),
TUB (HEX), and GAPDH (Quasar) assay set with an input of 100 ng–100 fg RNA was performed on a CFX96 Real-Time
PCR System. A, iTaq Universal Probes One-Step Kit exhibited the greatest dynamic range across all three assays,
which indicates a high degree of sensitivity. In addition, the iTaq Kit demonstrated the lowest standard deviations fortechnical replicates. B, TaqMan RNA-to-CT 1-Step Kit from Thermo Fisher Scientific did not perform as well, exhibiting 4logs of dynamic range for each assay (lower sensitivity) and higher standard deviations for technical replicates (lower
data precision). RFU, relative fluorescence units.

Amplification Reagents and Plastics

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27

4 PCR AND REAL-TIME PCR REAGENTS
iQ Supermixes

iQ Supermixes

iQ Multiplex Powermix
■

■

■

■

Robust supermix formulated for sensitive and efficient multiplex qPCR
 eliable quantification of up to 4 targets (expression levels can varyR
up to 106-fold between target genes) or up to 5 targets
 inearity over 6 orders of magnitude of input cDNA and 4 orders
Lof magnitude of input genomic DNA uitable for a wide variety of applications, including gene expression analysis,S
SNP genotyping, SNP analysis, GMO detection, and viral load detectioniQ SYBR® Green Supermix■■

 nalysis of low-, medium-, and high-abundance target genes with superiorA
sensitivity and efficiency
 ormulated for maximum SYBR® Green I stability and performanceF
in a wide variety of real-time PCR instruments

For more information, download
bulletins 2764 and 5348.

28

Accurate and Precise 5-Plex qPCR Results

RFU

iQ Supermixes contain hot-start iTaq DNA Polymerase, deoxynucleoside
triphosphates (dNTPs) qualified for qPCR, and buffer at concentrations optimalfor real-time PCR assays. These supermixes can be used across a wide dynamic
range of human genomic and plasmid DNA concentrations.

bio-rad.com

Amplification Reagents and Plastics

10 2

0

10		20			 30

40		

50		

CyclesDye

Efficiency, %Standard Curve, R 2

FAM
HEXTexas Red
Cy5
Quasar

97.8
98.9
99.7
98.1
99.8

1.000
1.000
1.000
1.000
1.000

iQ Multiplex Powermix produces highly accurate and precise 5-plex
qPCR results in a single well reaction across 6 logs of dynamic range.
RFU, relative fluorescence units.

PCR AND REAL-TIME PCR REAGENTS
Precision Melt Supermix

4

Precision Melt Supermix

■

■

■

■

■

■

 ensitive and specific discrimination of class I–IV SNPs across a broad
S
range of amplicons
Ideal solution for insertions or deletions >6 base pairs ccurate detection of the percentage of CpG methylamine statusA
for epigenetic studies
Ideal for mutation screening of small mutations or using a primer walking
approach for larger regions
 ost-saving application for upstream and downstream next-generation
C
sequencing prescreens and postvalidations
Exceptional room temperature stability for high-throughput HRM studies
Optimized formulation containing EvaGreen® Dye delivers robust PCR
and HRM performance

Applications and Uses
Genotyping■ Methylation studies■ Mutation detection■ Next-generation sequencing screening■ Species identification

A

B
0.02

0.02

0.00

0.00

–0.02

–0.02

–0.04

–0.04

–0.06

–0.06

–0.08

–0.08
75

76

77
78
79
Temperature, °C

75

80

76

77
78
79
Temperature, °C

80

Exceptional stability enables high-throughput genotyping analysis with Precision Melt Supermix.
Specific amplification and accurate discrimination of a class IV SNP (84 bp amplicon) from mouse
gDNA was performed on a CFX384 Real-Time PCR System either 0 hr (A) or 48 hr (B) after reaction
setup. Wild type (■), heterozygote ( ■), and homozygous mutant (■) are shown in the differenceplots normalized to wild-type samples. Total run time including melt curve = 150 min. RFU, relative
fluorescence units.
Accurate Methylation Detection
A

B
1.0

Normalized RFU■

0.8

Difference RFU■

Exceptional Stability

Difference RFU

Precision Melt Supermix is a superior high resolution melt (HRM) supermix
that delivers optimal discrimination for numerous applications.

0.6
0.4
0.2
0.0

Methylated DNA
■ 0%
■ 2%
■ 5%
■ 50%
■ 75%
■ 95%
■ 100%

0.0
–0.1
–0.2
–0.3
–0.4
–0.5
–0.6

74

76
78 80
82
Temperature, °C

84

74

76

78
80
82
Temperature, °C

84

Accurate methylation detection with Precision Melt Supermix. A, normalized melt curve; B, difference
curve. Mixtures of methylated and unmethylated human gDNA of varying ratios were analyzed using HRM
analysis on a CFX384 Real-Time PCR System. Increasing amounts of methylated DNA were analyzed formethylation of the human RARB2 gene. The genomic region contains seven CpG sites and is 88 bp inlength. Total run time including melt curve = 190 min. RFU, relative fluorescence units.

For more information,
download bulletin 6137.

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4 PCR AND REAL-TIME PCR REAGENTS
Precision Blue Real-Time PCR Dye

Precision Blue Real-Time PCR Dye
Precision Blue Real-Time PCR Dye is a concentrated, ready-to-use reagent that enhances
the visibility of real-time PCR reactions. This robust, versatile reagent is formulated for a wide
range of real-time PCR applications.
 acilitates precise pipetting and accurate reaction tracking when loading tubes or plates
F
Helps increase qPCR accuracy and reproducibility■ Decreases setup time without compromising qPCR data quality■ Useful for loading white or 384-well plates

Precision Blue Dye performs well and is compatible with:■ All PrimePCR Assays and Arrays■ Bio-Rad universal real-time supermixes and one-step kits
■ Fluorogenic probes and SYBR® Green chemistries■■

Precision Blue 200x Dye
Precision
Blue
Real-Time

Bio-Rad supermix
Supermix

Pipet
Sample DNA

1x qPCR
master mix

Pipetting a reaction mix into a PrimePCR Assay Plate.

30

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Amplification Reagents and Plastics

PrimePCR Assay PlatePCR AND REAL-TIME PCR REAGENTS
High-Fidelity and Standard PCR Reagents4High-Fidelity and Standard PCR Reagents
Bio-Rad offers reagents for both high-fidelity and standard PCR applications.
Use the selection guide to choose an appropriate DNA polymerase for yourapplication.
High-Fidelity and Standard PCR Reagents Selection Guide
iProof High-Fidelity
DNA Polymerase

iTaq
DNA Polymerase

Sso7d fusion enzyme technology

Yes

No

Hot-start enzyme activationNo

Yes, antibody-mediated5'➝ 3' exonuclease

No

Yes

3'➝ 5' exonuclease

Yes

No

Blunt or 3'-A ends

Blunt

3'-A

Fidelity (vs. Taq)

52x–

FeaturesLong amplification
Fast cycling

Yes, up to 37 kb

–

Yes

No

iProof High-Fidelity DNA Polymerase
iProof High-Fidelity DNA Polymerase is composed of a unique Pyrococcus-like
proofreading enzyme, which employs our patented* Sso7d fusion enzyme technology
for speed and accuracy. It is an ideal choice for applications that require high fidelity,
such as cloning. iProof DNA Polymerase features:■■■

High fidelity — iProof DNA Polymerase is 52-fold more accurate than Taq DNA
polymeraseSpeed — high processivity dramatically reduces extension time
(15–30 sec/kb) and overall reaction times
 uccessful amplification of long products with higher yields — fragments up toS
37 kb are amplified in less time and with less enzyme (0.25–1 U/reaction)
iProof DNA Polymerase is available in three convenient formats:
Convenient, ready-to-use 2x master mix
DNA polymerase kit, including reaction buffers■ PCR kit, including reaction buffers and controls■■

iTaq DNA Polymerase■■Antibody-mediatedhot-start DNA polymerase for quick 3 min activation
at 95°C olymerase prevents nonspecific amplification and primer-dimers in bothP
PCR and real-time PCR applications

For more information, download
bulletins 2779 and 5211.
* U.S. patents 6,627,424; 7,541,170; and 7,560,260.

Amplification Reagents and Plastics

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31

4 PCR AND REAL-TIME PCR REAGENTS
High-Fidelity and Standard PCR Reagents

High-Fidelity and Standard PCR Reagents
iProof High-Fidelity DNA Polymerase Performance
Amplification of Long Templates

Shorter Overall Reaction TimesA■ iProof High-Fidelity DNA Polymerase■ Common high-fidelity polymerase■ Taq polymerase

35 minBAC DNA (37 kb)

1 kb

2 hr

20

10

22

25

32

Template length

2 hr

37

1.5 hr8 kb

9.5 hr
5.5 hr5

2 hr 20 min
15 kb

16.5 hr(Failed amplification)
Total protocol timeB
Human genomic DNA (28 kb)

15

22

28

– 48 kb– 23 kb

7.9

3.8
2.9

iProof High-Fidelity DNA Polymerase amplifies long templates withhigh yields. A, various fragments up to 37 kb in length were amplified fromBAC DNA using a combined annealing/extension step of 10 min per cycle
and 30 U/ml of iProof Polymerase. B, various sequences up to 28.8 kb were
amplified directly from human genomic DNA using 30 U/ml of iProof Polymerase
in GC buffer with a combined annealing/extension time of 10 min per cycle.
BAC, bacterial artificial chromosome.

32

bio-rad.com

Amplification Reagents and Plastics

iProof High-Fidelity DNA Polymerase demonstrates unrivaled speed, leading todramatically shorter overall reaction times. The reaction protocol for iProof Polymerase
was compared to the recommended protocols for two competing polymerases. Eachprotocol was designed to amplify 1, 8, and 15 kb products in 30 cycles. Reactions with
iProof Polymerase used a two-step protocol with a combined annealing and extension
step, while the other reactions used three-step protocols with the minimum recommended
extension times. Overall reaction times include temperature ramping times.

PCR AND REAL-TIME PCR REAGENTS

Real-Time PCR Reagent Compatibility with Instruments

4Real-Time PCR Reagent Compatibility with Instruments
SYBR ® Green Supermixes

Probes Supermixes

One-Step RT-qPCR Supermix and Kits

SsoAdvanced
Universal SYBR®
Green Supermix

SsoAdvanced
Universal InhibitorTolerant SYBR®
Green Supermix

iTaq Universal
SYBR® Green
Supermix

iQ SYBR®
Green
SupermixiQ Multiplex
Powermix

SsoAdvanced
Universal
Probes Supermix

iTaq
Universal
ProbesSupermix

Reliance
One-Step
Multiplex
Supermix

iTaq Universal
SYBR® Green
One-Step Kit

iTaq Universal
Probes
One-Step Kit

CFX96, CFX96 Touch, CFX96
Touch Deep Well, CFX384,
CFX384 Touch, CFX Connect

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

iQ, iQ5, MyiQ, MyiQ2

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

MiniOpticon, DNA Engine
Opticon I and II

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

StepOne/StepOnePlus

✔

✔

✔

◆

◆

✔

✔

✔

✔

✔

7500, ViiA 7

✔

✔

✔

­—

­—

✔

✔

✔

✔

✔

7000, 7300, 7700, 7900HT,
QuantStudio Systems

✔

✔

✔

—

—

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

LightCycler 480, 96

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

LightCycler 1.0, 1.5, 2.0

▲

▲

▲

▲

▲

▲

▲

▲

▲

▲

LightScanner HR-1

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

LightScanner 32

▲

▲

▲

▲

▲

▲

▲

▲

▲

▲

Real-Time PCR Instrument

Bio-RadApplied Biosystems

Stratagene
Mx3000P, 3005P, 4000
Eppendorf
Mastercycler ep
realplex 2, 4
QIAGEN/Corbett
Rotor-Gene 3000,
6000, QRoche

BioFire✔ Recommended for use as is◆ ROX reference setting must be turned off▲ BSA must be added according to instrument specifications— Not compatible

Amplification Reagents and Plastics

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33

4 PCR AND REAL-TIME PCR REAGENTS
Ordering Information

Ordering Information
Catalog # Description
SsoAdvanced Universal Supermixes
1725270
SsoAdvanced Universal SYBR Green Supermix, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
1725271
SsoAdvanced Universal SYBR Green Supermix, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
1725272
SsoAdvanced Universal SYBR Green Supermix, 10 ml (10 x 1 ml vials),
1,000 x 20 μl reactions
1725274
SsoAdvanced Universal SYBR Green Supermix, 25 ml (5 x 5 ml vials),
2,500 x 20 μl reactions
1725275
SsoAdvanced Universal SYBR Green Supermix, 50 ml (10 x 5 ml vials),
5,000 x 20 μl reactions
1725280 SsoAdvanced Universal Probes Supermix, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
1725281
SsoAdvanced Universal Probes Supermix, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
1725282 SsoAdvanced Universal Probes Supermix, 10 ml (10 x 1 ml vials), 1,000 x 20 μl reactions
1725284 SsoAdvanced Universal Probes Supermix, 25 ml (5 x 5 ml vials), 2,500 x 20 μl reactions
1725285 SsoAdvanced Universal Probes Supermix, 50 ml (10 x 5 ml vials), 5,000 x 20 μl reactions
1725016
SsoAdvanced Universal Inhibitor-Tolerant SYBR Green Supermix, 2 ml (2 x 1 ml vials),
200 x 20 µl reactions
1725017
SsoAdvanced Universal Inhibitor-Tolerant SYBR Green Supermix, 5 ml (1 x 5 ml vial),
500 x 20 µl reactions
1725018
SsoAdvanced Universal Inhibitor-Tolerant SYBR Green Supermix, 10 ml (2 x 5 ml vials),
1,000 x 20 µl reactions

Visit bio-rad.com/RT-PCRreagentsfor additional real-time PCR reagents.

34

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Amplification Reagents and Plastics

Catalog #Description

iTaq Universal Supermixes
1725120
iTaq Universal SYBR Green Supermix, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
1725121
iTaq Universal SYBR Green Supermix, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
1725122
iTaq Universal SYBR Green Supermix, 10 ml (10 x 1 ml vials), 1,000 x 20 μl reactions
1725124
iTaq Universal SYBR Green Supermix, 25 ml (5 x 5 ml vials), 2,500 x 20 μl reactions
1725125
iTaq Universal SYBR Green Supermix, 50 ml (10 x 5 ml vials), 5,000 x 20 μl reactions
1725130
iTaq Universal Probes Supermix, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
1725131
iTaq Universal Probes Supermix, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
1725132
iTaq Universal Probes Supermix, 10 ml (10 x 1 ml vials), 1,000 x 20 μl reactions
1725134
iTaq Universal Probes Supermix, 25 ml (5 x 5 ml vials), 2,500 x 20 μl reactions
1725135
iTaq Universal Probes Supermix, 50 ml (10 x 5 ml vials), 5,000 x 20 μl reactions
Reliance One-Step Multiplex RT-qPCR Supermixes
12010176 Reliance One-Step Multiplex Supermix, 1 ml (1 x 1 ml vial), 200 x 20 μl reactions
12010220 Reliance One-Step Multiplex Supermix, 5 ml (5 x 1 ml vials), 1,000 x 20 μl reactions
12010221 Reliance One-Step Multiplex Supermix, 10 ml (2 x 5 ml vials), 2,000 x 20 μl reactions
iTaq Universal One-Step RT-qPCR Kits
1725150
iTaq Universal SYBR Green One-Step Kit, 1 ml (1 x 1 ml vial), 100 x 20 μl reactions
1725151
iTaq Universal SYBR Green One-Step Kit, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
1725140
iTaq Universal Probes One-Step Kit, 1 ml (1 x 1 ml vial), 100 x 20 μl reactions
1725141
iTaq Universal Probes One-Step Kit, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions

PCR AND REAL-TIME PCR REAGENTS

Ordering Information

Catalog #DescriptioniQ Supermixes
1725848 iQ Multiplex Powermix, 1.25 ml (1 x 1.25 ml vial), 50 x 50 μl reactions
1725849 iQ Multiplex Powermix, 5 ml (4 x 1.25 ml vials), 200 x 50 μl reactions
1708880 iQ SYBR Green Supermix, 2.5 ml (2 x 1.25 ml vials), 100 x 50 μl reactions
1708882 iQ SYBR Green Supermix, 12.5 ml (10 x 1.25 ml vials), 500 x 50 μl reactions
1708884 iQ SYBR Green Supermix, 25 ml (20 x 1.25 ml vials), 1,000 x 50 μl reactions
1708885 iQ SYBR Green Supermix, 50 ml (50 ml bottle), 2,000 x 50 μl reactions
1708886 iQ SYBR Green Supermix, 25 ml (5 x 5 ml vials), 1,000 x 50 μl reactions
1708887 iQ SYBR Green Supermix, 50 ml (10 x 5 ml vials), 2,000 x 50 μl reactions
1708860 iQ Supermix, 2.5 ml (2 x 1.25 ml vials), 100 x 50 μl reactions
1708862 iQ Supermix, 12.5 ml (10 x 1.25 ml vials), 500 x 50 μl reactions

Catalog #4

Description

Standard PCR Reagents
1725310
iProof HF Master Mix, 0.04 U/μl, 100 x 50 μl reactions
1725320
iProof GC Master Mix, 0.04 U/μl, 100 x 50 μl reactions
1725301
iProof High-Fidelity DNA Polymerase, includes 5x reaction buffers, MgCl2, and DMSO,
100 U, 2 U/μl1725302
iProof High-Fidelity DNA Polymerase, includes 5x reaction buffers, MgCl2, and DMSO,
500 U, 2 U/μl1725330
iProof High-Fidelity PCR Kit, includes 5x reaction buffers and DNA control, 50 U, 2 U/μl1725331
iProof High-Fidelity PCR Kit, includes 5x reaction buffers and DNA control, 200 U, 2 U/μl1708870
iTaq DNA Polymerase, 250 U, 5 U/μl1708875
iTaq DNA Polymerase, 5,000 U, 5 U/μl1725858
ROX Passive Reference Dye, 0.5 ml
1708874
dNTP Solution Mix, 200 μl
Additional Real-Time PCR Reagents
1725110
Precision Melt Supermix, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
1725112Precision Melt Supermix, 10 ml (10 x 1 ml vials), 1,000 x 20 μl reactions
1725555
Precision Blue Real-Time PCR Dye, 55 µl (1 x 1 ml vial), 500 x 20 μl reactions

Visit bio-rad.com/RT-PCRreagentsfor additional real-time PCR reagents.

Amplification Reagents and Plastics

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35

5 PREAMPLIFICATION
Preamplification is a powerful tool to enable highly
sensitive detection of a large number of targets
from a limited sample.
■

■

■

36

bio-rad.com

Amplification Reagents and Plastics

Proprietary buffers and Sso7d fusion technology generateunbiased preamplification reactions
Optimized for PrimePCR PreAmp Assays
Unparalleled sensitivity

PREAMPLIFICATION

Preamplification Selection Guide

5

Preamplification Selection Guide
Preamplification is a valuable tool that enables researchers to enrich specificgene targets of interest from a limited sample. Selecting a preamplification kit thatprovides unbiased preamplification is key to successful gene expression analysis.
Product
iScript Explore
One-Step RT and
PreAmp Kit

SsoAdvanced
PreAmp Supermix

Description
cDNA synthesis and preamplification
performed in a single step
■ RNA used as input sample■ Reverse transcription carried out
using gene-specific priming
cDNA synthesis and preamplification
performed in separate steps■ cDNA synthesis carried out using
any of the iScript family of reverse
transcription reagents■ cDNA used as input sample forpreamplification reaction■ Reverse transcription carried out
using gene-specific priming

Key Benefits
■
■
■

■

■
■

■

Unbiased preamplification
Protocol minimizes hands-on time
Compatible with PrimePCR PreAmp
Control
Kit integrates an effective gDNA
digestion step

Ideal Sample Types for Preamplification
FFPE samples
LCM samples■ Plant samples■ Rare samples

Single cells
Small biopsies■
Sorted cells■
Stem cells

■

■

■

■

Unbiased preamplification
Flexibility with cDNA synthesis
method of choice
cDNA can be archived for futurestudies of different genes

Amplification Reagents and Plastics

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37

5 PREAMPLIFICATION

iScript Explore One-Step RT and PreAmp Kit

iScript Explore One-Step RT and PreAmp Kit
iScript Explore One-Step RT and PreAmp Kit features:
1-step reverse transcription and preamplification — the first kit that eliminates
protocol steps by generating preamplified cDNA from RNA in a single step
■ Unbiased preamplification — can reliably profile panels of targets■ High sensitivity — enables reliable detection of genes expressed at low levels■ Preamplification control — preamplification efficacy can be easily assessed
when used with PrimePCR Long Noncoding RNA (lncRNA) Arrays or custom
PrimePCR Plates■ Effective genomic DNA removal — provides confidence that results are only for thetranscript of interest

Preamplification Does Not Introduce Bias
35
Cq without preamplification

■

30

25

20
R2 = 0.952
15
10

15
20
25
Cq with preamplification

30

High correlation between preamplified and non-preamplified samples. Potential for bias wasevaluated using ERCC RNA transcripts of a known copy number. ERCC concentrations from single
copy to 16,000 copies were used to assess bias across a wide range of transcript concentrations.
RNA samples were reverse transcribed using iScript Advanced cDNA Synthesis Kit for RT-qPCR
or reverse transcribed and preamplified using iScript Explore One-Step RT and PreAmp Kit. The
R2 value of 0.952 indicates a high correlation between samples with and without preamplification,
demonstrating that preamplification does not introduce bias. Cq, quantification cycle.

Visit bio-rad.com/iScriptExplore
for more information.

38

bio-rad.com

Amplification Reagents and Plastics

PREAMPLIFICATION

SsoAdvanced PreAmp Supermix

5

SsoAdvanced PreAmp Supermix
SsoAdvanced PreAmp Supermix provides unbiased, target-specificpreamplification from limited nucleic acids using up to 100 qPCR or
end-point assays and enabling more downstream reactions for screeningof respective targets.

This guide provides a comparison of PreAmp products.

Features of SsoAdvanced PreAmp Supermix
Unbiased preamplification for accurate expression analysis
from limited sample■
Requires as little as 100 pg of gDNA or cDNA■
Validated for SYBR® Green and probe chemistries■
Optimized for PrimePCR PreAmp Assays■
Improved performance using TaqMan and custom assays■
Superior sensitivity and efficiency■
Economically priced■

SsoAdvanced
PreAmp Supermix

TaqMan PreAmp
Master Mix(Thermo Fisher
Scientific)

PerfeCTa PreAmp
5x SuperMix(Quanta BioSciences)

Bias quantification score90% ≤ 0.75

90% ≤ 1.50

90% ≤ 1.50

Sensitive detectionof low-level target genes

•••

••

•

High efficiency, even fordifficult amplicons

•••

••

••

PrimePCR PreAmp,
TaqManTaqManUnknown

Property

Validated assays®

SYBR Green Assays✔

Probe assays

✔

✔

✔

50 x 50 µl

40 x 50 µl

40 x 50 µl

Number of reactions• Fair•• Good✔

••• Excellent

Ordering Information
Catalog #Description

iScript Explore One-Step RT and PreAmp Kits
12004856
iScript Explore One-Step RT and PreAmp Kit, 50 x 50 μl reactions
17002826
iScript Explore One-Step RT and PreAmp Kit, 250 x 50 μl reactions
SsoAdvanced PreAmp Supermix
1725160
SsoAdvanced PreAmp Supermix, 1.25 ml (1 x 1.25 ml vial), 50 x 50 μl reactions

Bias Quantification Score
A bias quantification (BQ) score provides a numerical value for the level of bias
observed in a preamplification reaction. It is based on comparing the theoreticaldifference between a sample with and without PreAmp compared to the observed(actual) difference between a sample with and without PreAmp. Zero indicatesno bias, and the farther from zero the BQ score becomes, the greater thebias introduced.

Visit bio-rad.com/PreAmp or
download or request bulletin 6576.

Amplification Reagents and Plastics

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39

6 PCR PLASTIC

CONSUMABLES

A large selection of PCR tubes, PCR plates, seals,
and accessories are precisely manufactured for
optimal fit and cycling performance in thermal
cyclers and real-time PCR systems.
■

■

■

■

40

bio-rad.com

Amplification Reagents and Plastics

Produced in Class 10,000 or 100,000 cleanroom environment
Certified to be free of DNase, RNase, and human gDNA
E xtremely uniform wells reduce well-to-well variability
in real-time PCR arp-free Hard-Shell Plates are designed for optimumW
performance with automation

PCR PLASTIC CONSUMABLES
Instrument Compatibility

6

Instrument Compatibility
Tubes
Individual High-Profile

Strips High-Profile

Strips Low-Profile

TBI0201, TFI0201, TWI0201

TBCxxxx, TBSxxxx

TLSxxxx

Bio-Rad C1000, C1000 Touch, S1000

✔

▲

✔

Bio-Rad DNA Engine,
DNA Engine Tetrad,
DNA Engine Tetrad 2,
DNA Engine Dyad,
Dyad Disciple, PTC-100

✔

▲

✔

Bio-Rad T100, MyCycler✔

✔

Bio-Rad iCycler✔

✔

Bio-Rad MJ Mini✔

▲

Applied Biosystems 0.2 ml Tube Cyclers(2720, 9700, Veriti)✔

✔

Catalog #Thermal Cycler

Applied Biosystems 0.1 ml Tube Cyclers(9800 Fast, Veriti Fast)
Eppendorf Mastercycler Series

✔

✔
✔

▲

✔

Real-Time PCR Instrument
CFX Opus 96, CFX Opus 384✔

Bio-Rad CFX96, CFX96 Touch, CFX96 Touch
Deep Well, CFX Connect✔

Bio-Rad iCycler iQ, iQ5, MyiQ, MyiQ2✔

Bio-Rad Chromo4▲✔

Bio-Rad DNA Engine Opticon, Opticon 2✔

Bio-Rad MiniOpticon*✔

Applied Biosystems Standard Systems(7300, 7500, 7900HT)✔

Applied Biosystems Fast Systems (7500 Fast,
7900HT Fast, StepOne, StepOnePlus)✔

Eppendorf Mastercycler ep realplex▲

Stratagene (Agilent) Mx Series✔

QIAGEN/Corbett Rotor-Gene✔ Recommended✔

✔

▲ Compatible

* The MiniOpticon Real-Time PCR System is factory calibrated for white tubes and white-well plates. White plastics are recommended due to their superior signal-to-noise ratio. Using clear tubes or clear-well plates
on this instrument will require user calibration.

Amplification Reagents and Plastics

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41

6 PCR PLASTIC CONSUMABLES
Instrument Compatibility

96- and 48-Well Plates

Catalog #384-Well Plates

Hard-Shell
Skirted
Low-Profile

Hard-Shell
Semi-Skirted
High-Profile

Hard-Shell
Semi-Skirted
Low-Profile

Hard-Shell
480

Multiplate
Unskirted
High-Profile

Multiplate
Unskirted
Low-ProfileiQ
Semi-Skirted
High-Profile

Hard-Shell
Standard

Hard-Shell
480

HSP9xxx

HSS9xxx

HSL9xxx

HSR99xx

MLPxxxx

MLLxxxx

2239441

HSP3xxx

HSR48xx

✔

▲

▲

▲

▲

▲

✔

▲

✔

▲

✔

▲

✔

▲

Thermal Cycler
Bio-Rad C1000, C1000 Touch,
S1000
Bio-Rad T100
Bio-Rad DNA Engine, DNA Engine Tetrad,
DNA Engine Tetrad 2, DNA Engine Dyad

✔

✔

▲

▲

▲

✔

Bio-Rad iCycler, MyCycler
Bio-Rad MJ Mini▲✔

Applied Biosystems 0.1 ml Tube Cyclers(9800 Fast, Veriti Fast )▲✔

Except MyCycler

Applied Biosystems 0.2 ml Tube Cyclers(2720, 9700, Veriti)

▲

▲
✔

▲
✔

▲

▲
✔

Applied Biosystems 384-Well Cyclers(9700, Veriti)
Eppendorf Mastercycler Series

✔

▲

▲

▲

▲

▲

Real-Time PCR Instrument
Bio-Rad CFX Opus 96, CFX Opus 384✔

▲

Bio-Rad CFX96, CFX96 Touch,
CFX Connect, CFX384,* CFX384 Touch*✔

▲

Bio-Rad CFX96 Touch Deep Well✔

Bio-Rad iCycler iQ, iQ5, MyiQ, MyiQ2
Bio-Rad DNA Engine Opticon,
Opticon 2, MiniOpticon*

▲

▲

▲

▲

✔

▲

✔

▲

✔

▲

✔
▲

Except MiniOpticon

✔

▲

▲

Except 7900HT,

Except 7900HT,

QuantStudio

QuantStudio✔

Applied Biosystems Fast Systems(7500 Fast, 7900HT Fast, StepOne, StepOnePlus,
QuantStudio Systems, ViiA 7 Fast)

Except StepOne,▲

StepOnePlus,

Except 7900HT Fast

7900HT Fast✔

Stratagene (Agilent) Mx Series
Roche LightCycler 96, 480✔ Recommended▲✔

Applied Biosystems Standard Systems(7300, 7500, 7900HT, QuantStudio Systems, ViiA 7)

Eppendorf Mastercycler ep realplex

✔

▲

▲

▲

✔

▲
✔

▲

▲
▲
✔

▲ Compatible

* C FX384, CFX384 Touch, and MiniOpticon Real-Time PCR Systems are factory calibrated for white tubes and white-well plates. White plastics are recommended due to their superior signal-to-noise ratio. Using clear tubes or clear-well plates 		
on these instruments will require user calibration.
Contact Bio-Rad technical support for compatibility with additional instruments.

42

bio-rad.com

Amplification Reagents and Plastics

Go to bio-rad.com/PCRPlasticSelector anduse our interactive selection tool to to find the
PCR plastics that fit your instrument.

PCR PLASTIC CONSUMABLESPCR Tubes and Strips

6

PCR Tubes and Strips
0.2 and 0.5 ml Individual PCR Tubes

Low-Profile PCR Tube Strips

 igh-profile PCR tubes with double-locking capsH
for a stronger seal during cycling■ Flat, frosted caps are easy to label■ 
Options with attached caps for greater convenienceand lower risk of contamination■

High-Profile PCR Tube Strips

Lower height reduces the potential for condensation
Designed to allow greater light capture influorescence assays■ Opaque white color option maximizes detection signal■ Maximum volume: 200 µl■■

Flat and Domed Cap Strips for PCR Tubes and PCR PlatesThin-walled tubes for superior heat transfer
8- and 12-tube strips for use on 48- or 96-wellsample blocks■ Available in a variety of colors for easy
sample tracking■ Maximum volume: 300 µl

Ultraclear flat cap strips are ideal for qPCR
Designed for extremely tight sealing for thermal cycling
and storage (–20 and 4˚C)
■ Flat caps have optimal light transmittance for real-time
PCR on PCR tubes or plates■ Great option to save plates if fewer wells areneeded during PCR

■

■

■

■

ECT2000
Strip Cap Tool

TLS0801, clear
TLS0851, white
Low-Profile Tube Strips without Caps

TBS0201, 8-tube, clear
TBS1201, 12-tube, clear
High-Profile Tube Strips without Caps

TCS0803, ultraclear
Optical Flat 8-Cap Strips

TCS0801, 8-cap, clear
TCS1201, 12-cap, clear
Domed Cap Strips

Amplification Reagents and Plastics

bio-rad.com

43

6 PCR PLASTIC CONSUMABLES
Hard-Shell PCR Plates

Hard-Shell PCR Plates
Hard-Shell Technology
The skirt and deck of a
Hard-Shell Plate prevent
warping and shrinkage

The patented two-component design of Hard-Shell Plates is specificallyengineered to withstand the stresses of thermal cycling. Benefits include: uperior stability and flatness allow precise positioning and robotic handlingS
Sturdy plate design is ideal for heat sealing methods■ 
Warp-resistant feature provides durability during automation, high-speed
centrifugation, and storage (even –80°C)■ 
User-readable barcode options for convenient sample tracking in
high-throughput settings■ Black alphanumeric labeling for easy well identification■ 
Footprint and well spacing designed to match ANSI/SBS
standard dimensions■ 
Composition helps prevent DNA binding■ Polypropylene resin allows superior well-to-well uniformity
for reliable and reproducible real-time qPCR results

Raised rims allow
for tight sealing

Rigid skirt ideal
for heat sealing■■Thin-wall polypropylene V-shaped
wells enable optimal thermal transfer
and recovery of low-volume samples

Enhanced Real-Time PCR Sensitivity
Relative signal strength

3.0
2.5
2.0
1.5
1.0
0.5
0
HSP9955, white shell, white well, barcoded
Hard-Shell Low-Profile 96-Well Skirted PCR Plates
Other colors and barcode option are available.

Clear

White

Black

For more information,
download bulletin 5496.

44

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Amplification Reagents and Plastics

PCR PLASTIC CONSUMABLES
Hard-Shell PCR Plates

6

Hard-Shell PCR Plates

15.51 mm
Low-profile

9.90 mm

HSL9645, green shell, white wellHard-Shell Low-Profile 96-Well
Semi-Skirted PCR Plates

Semi-skirt

HSP3821, yellow shell, clear wellHard-Shell 384-Well
Skirted PCR Plates

20.75 mm
High-profile

15.51 mm
Low-profile

Semi-skirt

HSR9905, clear shell, white wellHard-Shell 96-Well
480 PCR Plates

Semi-skirt

HSS9665, black shell, white wellHard-Shell High-Profile 96-Well
Semi-Skirted PCR Plates

For more information,
download bulletin 5496.

Amplification Reagents and Plastics

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45

6 PCR PLASTIC CONSUMABLESPCR Plates

PCR Plates
Multiplate 96-Well and 48-Well Unskirted PCR Plates ingle-component polypropylene allows low protein binding and excellent
Ssample retention■ High-profile (20.7 mm) and low-profile (15.5 mm) options allow broad real-time
PCR instrument compatibility■ White-well option for maximizing real-time PCR detection sensitivity■ 
Designed to allow easy cutting with scissors (when less than a full plateis needed)■

MLP4801, clear
Multiplate High-Profile 48-Well
Unskirted PCR Plates

iQ High-Profile 96-Well Semi-Skirted PCR Plates emi-skirt design provides a labeling surface for easy sample tracking
S
Composition designed to provide stiffness during plate handling■ High-profile (20.7 mm) plate has perforations every 3 columns
for convenience (when less than a full plate is needed)■■

MLL4801, clear
MLL4851, white
Multiplate Low-Profile 48-Well
Unskirted PCR Plates

MLL9601, clear
Multiplate Low-Profile 96-Well
Unskirted PCR Plates

2239441iQ High-Profile 96-Well
Semi-Skirted PCR Plates46

bio-rad.com

Amplification Reagents and Plastics

PCR PLASTIC CONSUMABLESPCR Plate Sealing

6

PCR Plate Sealing
PX1 PCR Plate Sealer

Seals Validated for PX1 PCR Plate Sealer

The PX1 PCR Plate Sealer is a semiautomated heat sealer for consistentand uniform sealing across an entire microplate. Features include:

Optically Clear Heat SealIntuitive touch-screen user interface for extreme convenienceand ease of use■ Programmable sealing protocols for quick access■ Small footprint suitable for crowded laboratory benches■ Compatible with a variety of heat sealing films and foils
and a wide range of PCR plates■■Ideal for real-time PCR
Excellent optical clarity■ 
Peelable for easy sample retrieval■ Compatible with PCR■

Permanent Clear Heat Seal

Ideal for water bath cycling
Nonpeelable, nonpierceable seal
■ High solvent resistance■■

Pierceable Foil Heat Seal ully validated for the QX200
F
Droplet Digital PCR System workflow■ 
Easily pierceable with a pipet tip■ 
User friendly — colored stripe clearly identifies
sealing surface■ Compatible with PCR■

Peelable Foil Heat Seal

Ideal for low-temperature sample storage
Can be easily peeled from microplates stored
in a –80°C freezer or in liquid nitrogen■ Compatible with PCR■■

For more information, download
or request bulletin 6257.

Amplification Reagents and Plastics

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47

6 PCR PLASTIC CONSUMABLESPCR Plate Sealing

Microseal 'F' Foil

Microseal 'B' Adhesive Seals, Optically Clear trongest adhesive-based optically clear sealing option
S
designed for real-time PCR plates■ Withstands multiple storage or transport temperatures(–40 to 110°C)■

 luminum foil allows opaque sealing option for DNA sequencing
A(ABI 3700 DNA Analyzer) and sample storage■ 
Acts as a barrier against evaporation in extreme temperatures(–80 to 105°C)■■

Pierceable foil for easy sample retrieval

Microseal 'C' Optical Seals
 ptically clear adhesive films designed for tight sealsOeven with wells with raised rims
■ Pressure-sensitive adhesive allows easy application
during plate sealing■ Designed with superior optical properties
for real-time PCR■

MSB1001
Microseal 'B' Adhesive Seals

MSC1001
Microseal 'C' Optical Seals

Ordering Information
Catalog # Description
PCR Plate Sealing
1814000
1814030

PX1 PCR Plate Sealer, includes heat sealing instrument, 96-well/384-well plate support block,sealing frame, power cord
Optically Clear Heat Seal, 100 seals

1814035

Permanent Clear Heat Seal, 100 seals

1814040

Pierceable Foil Heat Seal, 100 seals

1814045

Peelable Foil Heat Seal, 100 seals

MSA5001 Microseal 'A' Film, 50 seals
MSB1001 Microseal 'B' Adhesive Seals, optically clear, 100 seals
MSC1001 Microseal 'C' Optical Seals, 100 seals
MSF1001 Microseal 'F' Foil, 100 seals
MSR0001 Sealing Roller, for film seals48

bio-rad.com

Amplification Reagents and Plastics

Microseal 'A' Film nonoptical, nonadhesive sealing option for quick pressure-basedAsealing of plates■ 
Allows easy removal without the risk of aerosol formation, minimizing
cross-contamination■ Convenient option for standard PCR needs■

MSA5001
Microseal 'A' Film

MSF1001
Microseal 'F' Foil

MSR0001
Sealing Roller

PCR PLASTIC CONSUMABLES
Ordering Information

6

Ordering Information
Catalog #Description

Description

Clear Well

White Well

Black Well

HSP9601

HSP9655—

HSP9611——

Yellow shell

HSP9621——

Blue shell

HSP9631

HSP9635—

PCR Tube Strips

Green shell

HSP9641

HSP9645

TBS0201

8-Tube Strips without Caps, clear, 120 strips (960 PCR tubes)

TBS1201
TBC0802

TLS0801

12-Tube Strips without Caps, clear, 100 strips (1,200 PCR tubes)
8-Tube Strips and Domed Cap Strips, clear, 20 bags of 12 x 8-tube strips
and 12 x 8-cap strips (1,920 PCR tubes and 1,920 caps)
12-Tube Strips and Domed Cap Strips, clear, 20 bags of 8 x 12-tube strips
and 8 x 12-cap strips (1,920 PCR tubes and 1,920 caps)
Low-Profile 8-Tube Strips without Caps, clear, 120 (960 PCR tubes)

Black shell
HSP9661
HSP9665
White shell, barcoded
HSP9901
HSP9955(Row H and Column 12)
Hard-Shell High-Profile 96-Well Semi-Skirted PCR Plates (25 plates)

TLS0851

Low-Profile 8-Tube Strips without Caps, white, 120 (960 PCR tubes)

Individual PCR Tubes

Hard-Shell Plates

TFI0201

PCR Tubes with Flat Caps (0.2 ml), clear, 1,000

Hard-Shell Low-Profile 96-Well Skirted PCR Plates (50 plates)

TWI0201

PCR Tubes with Domed Caps (0.2 ml), clear, 1,000

White shell

TBI0201

PCR Tubes without Caps (0.2 ml), clear, 1,000

Red shell

TBI0501

PCR Tubes with Flat Caps (0.5 ml), clear, 1,000 (2 bags of 500)

TBI0502

PCR Tubes with Flat Caps (0.5 ml), clear, 800 (8 bags of 100)

TBC1202—
HSP9666—

Clear shell

HSS9601——

Green shell

HSS9641——

TCS0801

Domed 8-Cap Strips, for PCR tubes and plates, clear, 120

Black shell—
HSS9665
Clear shell, barcoded
HSS9901—(Row H, Column 1, and Column 12)
Hard-Shell Low-Profile 96-Well Semi-Skirted PCR Plates (25 plates)

TCS1201

Domed 12-Cap Strips, for PCR tubes and plates, clear, 200

Clear shell

HSL9605—

TCS0803

Optical Flat 8-Cap Strips, for PCR tubes and plates, ultraclear, 120

Green shell
HSL9641
Clear shell, barcoded
HSL9901(Row H and Column 12)
Hard-Shell 384-Well Standard PCR Plates (50 plates)
Clear shell
HSP3801
Red shell
HSP3811
Yellow shell
HSP3821
Blue shell
HSP3831
Green shell
HSP3841
Black shell—
Clear shell, barcoded
HSP3901(Row P and Column 24)

HSL9645
HSL9905——

HSP3805————
HSP3865
HSP3905—————
HSP3866—

Description

Number of Plates

Number of Plates and Seals

25 Plates

100 Plates, 100
Microseal 'C' Seals

Cap Strips

Capping Tools and Racks
TRC9601
TRC0501
ECT1000
ECT2000

PCR Tube Rack, ANSI/SBS standard, white, 10
96-Place Racks, with covers, for PCR tubes and unskirted and semi-skirted
microplates, assorted colors, 5
Easy Cap Tool, ensures tight seal for 0.2 ml PCR tubes
or 96-well microplates
Strip Cap Tool, for sealing 8- and 12-cap strips on PCR plates or tubes

PCR Plates
Multiplate 48-Well PCR Plates
MLP4801

Multiplate High-Profile 48-Well Unskirted PCR Plates, clear, 50 plates

MLL4801

Multiplate Low-Profile 48-Well Unskirted PCR Plates, clear, 50 plates

MLL4851

Multiplate Low-Profile 48-Well Unskirted PCR Plates, white, 50 plates

HSL9601

Multiplate High-Profile 96-Well Unskirted PCR Plates
MLP9601

Multiplate High-Profile 96-Well Unskirted PCR Plates, clear, 25 plates

MLP9651

Multiplate High-Profile 96-Well Unskirted PCR Plates, white, 25 plates

MLP9631

Multiplate High-Profile 96-Well Unskirted PCR Plates, blue, 25 plates

Multiplate Low-Profile 96-Well Unskirted PCR Plates
MLL9601

Multiplate Low-Profile 96-Well Unskirted PCR Plates, clear, 25 plates

MLL9651

Multiplate Low-Profile 96-Well Unskirted PCR Plates, white, 25 plates

iQ High-Profile 96-Well Semi-Skirted PCR Plates
2239441iQ High-Profile 96-Well Semi-Skirted PCR Plates, 25 plates

Hard-Shell 96-Well 480 PCR Plates(with barcode on Row A side)
Clear shell, white well
Clear shell, clear well

HSR9905
HSR9901——50 Plates

HSR9905K
HSR9901K
100 Plates, 100
Microseal 'C' Seals

Hard-Shell 384-Well 480 PCR Plates(with barcode on Row A and Column 24 side)
Clear shell, white well

HSR4805

HSR4805K

Clear shell, clear well

HSR4801

HSR4801K

Amplification Reagents and Plastics

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49

7 PrimePCR ASSAYSAND PANELS

PrimePCR Assays and Panels for real-time
PCR are expertly designed and are guaranteed
to perform.
■

■

■

■

50

bio-rad.com

Amplification Reagents and Plastics

E xpertly designed SYBR® Green and probe assays for qPCR,
preamplification, and Droplet Digital PCR
Wet-lab validated for guaranteed performance; eachmRNA assay for the human, mouse, and rat genomes
was experimentally tested for optimal efficiency, specificity,
sensitivity, and linear dynamic range
Assays available for expression analysis of messenger
RNA and long noncoding RNA targets
Compliant with the MIQE guidelines

PrimePCR ASSAYS AND PANELSHigh-Performance Assay Solutions

7

High-Performance Assay Solutions

Guaranteed Performance

Wide Range of Predesigned Diseaseand Pathway-Specific Panels

Offers wet-lab validation of every
mRNA assay for human, mouse,
and rat genomes■ Eliminates time-consuming
optimization■ 
A ids in MIQE compliance

Complete Solution for Real-Time PCR xpertly curated predesigned arraysE
to include the most biologically
relevant gene targets■ 
Customizable arrays to selecttargets of interest■ 
Integrated with CFX Maestro Software
for simple setup and analysis■■

Aurum Total RNA Kits
iScript cDNA Synthesis Kits
■ Supermixes for qPCR
■ Preamplification reagent kits
and PrimePCR PreAmp Assays■ PCR plates and tubes■ Real-time PCR instruments■ CFX Maestro Software■ PrimePCR Analysis Software■■

Assay Design
Advantages■

Assay specificity
confirmed by
next-generation
sequencing■

Avoided common
single nucleotide
polymorphisms
in target regions■

Designed intronspanning assays
whenever possible■

Avoided secondary
structures in primer
annealing sites■

Maximized fractionof transcript isoforms
being detected■Compatible withstandard assay
conditions

Amplification Reagents and Plastics

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51

7 PrimePCR ASSAYS AND PANELS
PrimePCR Real-Time PCR Products

PrimePCR Real-Time PCR Products
Predesigned SYBR® Green Assays

Predesigned Pathway Panels

Transcriptome-wide primer assays for SYBR ® Green
gene expression analysis are available in 200, 1,000,
or 2,500 reaction sizes.

A large selection of predesigned
disease- and pathway-specificpanels are available.

Predesigned Probe Assays
Transcriptome-wide probe assays for gene expression
analysis are available in 500, 1,000, or 2,500
reaction sizes with five fluorophore options.
Custom Assays

Custom PCR Plates
Thousands of PrimePCR Assays areavailable to create custom-configured
96- and 384-well PCR plates, which canbe ordered with SYBR® Green Assays.

User-defined primer and probe
sequences can be ordered.
PrimePCR Lookup Tool
PreAmp Assays
Concentrated primers are available fortarget-specific preamplification of limited
nucleic acid samples.

Quickly find your gene targets.
Visit bio-rad.com/PrimePCR to view our fulloffering of high-performance PrimePCR Assays.

DNA Templates
Gene-specific synthetic DNA templates are designed
to serve as a positive control when used with thecorresponding gene assay.
Experimental Controls
Control assays can be used to assess reverse
transcription, RNA quality, gDNA contamination,
and PCR performance.

52

bio-rad.com

Amplification Reagents and Plastics

Visit bio-rad.com/PrimePCR or
download bulletins 6290, 6512,
and 6595 for more information.

PrimePCR ASSAYS AND PANELSAssay Performance Standards and Validation

7

Assay Performance Standards and Validation
Assay Performance Standards

Assay Validation

Standard

ResultSensitivity

Accurate detection of 20 copies

Specificity

Validated amplicon sequence with next-generation
sequencing; minimal primer-dimer formation and
gDNA cross-reactivity

Amplification efficiency

90–110%Linear dynamic range

Minimum of 6 orders of magnitude; detection of a synthetic
template standard curve from 20 to 20,000,000 copies

R2

>0.98

PrimePCR Assays for the human, mouse, and rat genomes were validated using iScript
Advanced cDNA Synthesis Kit for RT-qPCR and SsoAdvanced SYBR® Green Supermix on
an automated CFX384 Touch Real-Time PCR System.

™

Amplification Reagents and Plastics

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53

7 PrimePCR ASSAYS AND PANELSPredesigned mRNA Pathway Panels

Predesigned mRNA Pathway Panels
The Most Focused Approach for Real-Time PCR
Bio-Rad collaborated with Thomson Reuters to expertly design
an extensive range of predesigned panels. Each real-time
PCR plate contains the most biologically relevant gene targets
in a canonical pathway, disease, or biological process.
■■Obtain complete pathway data from a single experiment
Gain new insights using integrated data analysis tools

Pathway Panels
Pathway-Focused Analysis

PrimePCR Pathway Panels were designed for morethan 300 canonical pathways. Pathway panels enable
complete pathway analysis for differentially expressed,
top-ranked gene targets.

Collection Panels
Broad Target Exploration

PrimePCR Collection Panels represent the top-ranked
gene targets for differential gene expression analysis,
allowing for a more general survey of gene targets
across a biological process or group.

Target Ranking
Gene assays present on predesigned panels havebeen prioritized based on three main criteria:

Disease Panels ow often a gene changes expression level inHtranscriptome studies■ 
How much attention was paid to this gene in theoverall scientific research■ How interesting the scientific community foundthis gene in the last 2 years

PrimePCR Disease State Panels were designed byreferencing the National Library of Medicine Medical
Subject Headings (MeSH) database. Disease statepanels allow for the thorough investigation of previously
published, differentially expressed genes withina specified pathology.

■54

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Amplification Reagents and Plastics

Disease-Focused Analysis

PrimePCR ASSAYS AND PANELS

PrimePCR Panels for a Broad Range of Pathways and Disease States

7

PrimePCR Panels for a Broad Range of Pathways and Disease States
DiseasesBacterial infections and fungal mycoses
Cancer and neoplasms■ Cardiac hypertrophy■ Cardiovascular diseases■ Congenital, hereditary, and neonatal
diseases and abnormalities■ Cystic fibrosis■ Digestive system diseases■ Endocrine system diseases■ Eye diseases■ Female urogenital diseases andpregnancy complications■ Hemic and lymphatic diseases■ Immune system diseases■ Male urogenital diseases■ Mental disorders■ Musculoskeletal diseases■ Nervous system diseases■ Nutritional and metabolic diseases■ Otorhinolaryngologic diseases■ Parasitic diseases■ Pathological conditions,signs, and symptoms■ Respiratory tract diseases■ Skin and connective
tissue diseases■ Stomatognathic diseases■ Viral diseases■ Wounds and injuries

Processes
Apoptosis and survival
Blood coagulation■ Cell adhesion■ Cell cycle■ Chemotaxis■ Cytoskeleton remodeling■ Development■ DNA damage■ Hypoxia response■ Immune response■ Muscle contraction■ Neurophysiological process■ Oxidative stress■ Proteolysis■ Reproduction■ Transcription■ Translation■ Transport

Metabolism
Amino acid metabolism
Carbohydrate metabolism■ Lipid metabolism■ Nucleotide metabolism■ Regulation of lipid metabolism■ Regulation of metabolism■ Steroid metabolism■ Vitamin and cofactor metabolism■ Xenobiotic metabolism

Protein Function
Cytokines and chemokines
G proteins■ Growth factors■ Hormones■ Kinases■ Phosphatases■ Second messengers■ Transcription factors

■

■

■

■

■

■

■

■

More than 1,000 unique panels are available
in these categories. Visit bio-rad.com/
biorad.com/
PrimePCRpanels for more information.

Amplification Reagents and Plastics

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55

7 PrimePCR ASSAYS AND PANELSPredesigned and Custom PrimePCR Long Noncoding RNA (lncRNA) Arrays

Predesigned and Custom PrimePCR Long Noncoding
RNA (lncRNA) Arrays
Simplified Long Noncoding RNA Discovery

Create Your Own Long Noncoding RNA Array

Predesigned PrimePCR lncRNA qPCR Arrays facilitate lncRNA discovery byoffering panels associated with specific research areas. Each plate was curated toinclude a combination of frequently cited lncRNAs as well as novel lncRNAs to aid
in target discovery.

Custom PrimePCR lncRNA qPCR Arrays simplify lncRNA expression research.
Easily expand upon next-generation sequencing and literature data mining projects
by using qPCR arrays tailored to your specific research question. Our custom
qPCR arrays help move your research forward by providing a well-accepted, highly
sensitive, and cost-effective approach to gene expression research. Custom qPCR
arrays enable you to go from RNA sample through data analysis in a single day.

 aster preamplification setup with matching PrimePCR lncRNA PreAmp PoolsFE xtensively wet-lab validated to ensure stringent performance standards are
met by lncRNA assays on predesigned plates■ Great companion for predesigned PrimePCR mRNA Arrays■■

IMMUNOLOGY56

bio-rad.com

ATHEROSCLEROSISEPIGENETICS
CELL CYCLEECM AND ADHESIONGROWTH FACTORSHYPOXIASTEM CELLSCYTOKINES
INNATE/ADAPTIVE IMMUNE RESPONSE
INTERFERON a/b
INFLAMMATION
TGF-b SIGNALING

Amplification Reagents and Plastics

Assays for lncRNA

CANCER

CELL PATHWAYS

Available lncRNA Arrays hoose from more than 23,000 lncRNAs to customize array plates withC
only your targets of interest■ 
Simultaneously examine both lncRNA and mRNA expression patterns■ 
Design and include your own PCR primers on any custom plate■

Assays for mRNA

GENERAL CANCER
BRAIN CANCERBLOOD CANCERSLUNG CANCER
COLORECTAL CANCER
BREAST CANCERPROSTATE CANCERANGIOGENESIS

PrimePCR ASSAYS AND PANELS
PrimePCR Long Noncoding RNA Assays

7

PrimePCR Long Noncoding RNA Assays
Assay Formats to Fit Your Needs

■

■

■

■

lncRNA SYBR® Green Primer AssayslncRNA Probes Assays compatible with multiplexing. Available reporter dyes:
FAM, HEX, Texas Red 615, Cy5, and Cy5.5 oncentrated PreAmp Assays that can be used for both preamplification andCdownstream qPCR reactions

DNA assay templates for every human lncRNA assay serve as a positive control

PrimePCR lncRNA Assays are also compatible with Droplet Digital PCR workflows.

Amplification Reagents and Plastics

bio-rad.com

57

7 PrimePCR ASSAYS AND PANELS

Experimental Controls and Reference Gene Assays

Experimental Controls and Reference Gene Assays
Controls
Experimental control assays and synthetic DNA templates are designed to assess
1
9
17
25
33
41
49
57
65
the key experimental factors that may impact your real-time PCR results.

1

9

17

25

73

81

89 1
PAQ
PAQ
90/Ref2

2

10

18

26

34

42

50

58

66

74

82

1

9

17

25

33

41

49

57

65

73

81

89

3

11

19

49

27
57
26

35
65
34

43
73
42

51
81
50

59
89/Ref
58

67

75

83

91/Ref

2

33

10

41

18

66

74

82

90/Ref

129
42
119

2017
50
1917

2825
58
2725

33
36
66
33
35

41
44
74
41
43

49
52
82
49
51

57
60
PAQ
57 1
59

65
68
65
67

73
76
73
75

81
84
81
83

89
gDNA
89
91/Ref

2

10

18

26

41
34
31

3

11

19

27

52
35
42

1310
43
1210

2118
51
2018

2926
59
2826

34
37
67
34
36

42
45
75
42
44

50
53
83
50
52

58
61
PAQ
58 2
60

66
69
66
68

74
77
74
76

82
85
82
84

90/RefPCR
90/Ref
gDNA

4

12

20

28

3
336

11
14
44
11
13

19
22
52
19
21

27
30
60
27
29

35
38
68
35
37

43
46
76
43
45

51
54
84
51
53

59
62
gDNA
59
61

67
70
67
69

75
78
75
77

83
86
83
85

91/Ref
RQ 1
91/Ref
PCR

5

13

21

29

4
437

12
15
45
12
14

20
23
53
20
22

28
31
61
28
30

36
39
69
36
38

44
47
77
44
46

52
55
85
52
54

60
63
PCR
60
62

68
71
68
70

76
79
76
78

84
87
84
86

gDNA
RQ 2
gDNA
RQ 1

6

14

22

30

5
538

13
16
46
13
15

21
24
54
21
23

29
32
62
29
31

37
40
70
37
39

45
48
78
45
47

53
56
86
53
55

61
64
RQ
61 1
63

69
72
69
71

77
80
77
79

85
88
85
87

PCR
RT
PCR
RQ 2

6
639

22
55
22
24
23
56
23

30
63
30
32
31
64
31

38
71
38
40
39
72
39

46
79
46
48
47
80
47

54
87
54
56
55
88/Ref
55

62
RQ
62 2
64
63
RT
63

70

78

86

RQ 1

7
740

14
47
14
16
15
48
15

70
72
71

78
80
79

86
88
87

RQ 1
RT
RQ 2

71

79

87

RQ 2

8

16

24

32

40

48

56

64

72

80

88

RT

8

16

24

32

40

48

56

64

72

80

88

RT

7

15

23

31

8

16

24

32

6

5
7

6
8

7

8

Reference Gene Assays
We have suggested a set of commonly used reference genes that can be used individuallyor easily screened using our preplated 96-well and 384-well reference gene panels.
Reference gene assays may also be added to custom-designed plates.

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Amplification Reagents and Plastics

Preamplification Quality Assay
The PrimePCR Preamplification Quality Assay isdesigned to work with iScript Explore One-Step
RT and PreAmp Kit to determine the efficiency of
preamplification.
DNA Contamination Control Assay
The PrimePCR DNA Contamination Control Assay
is designed to determine if gDNA is present ina sample at a level that may affect PCR results.
Positive PCR Control Assay
The PrimePCR Positive Control Assay is designed
to assess how a given experimental sample may
adversely affect PCR performance.
RNA Quality Assay
The PrimePCR RNA Quality Assay is designed
to determine whether RNA integrity may
adversely affect PCR results.
Reverse Transcription Control Assay
The PrimePCR Reverse Transcription Control Assay
is designed to qualitatively assess the performanceof the RT reaction.

PrimePCR ASSAYS AND PANELS
PrimePCR Data Analysis

7

PrimePCR Data Analysis
CFX Maestro Software
Ordering a PrimePCR Plate or Assay is just the beginning of a seamless and integrated
workflow, from reaction setup to data acquisition and analysis using CFX Maestro Softwareand PrimePCR Analysis Software. The expert design and wet-lab validation of PrimePCR
Assays for the human, mouse, and rat genomes ensure optimal assay performance sothat time once spent optimizing runs can now be devoted to analyzing and interpreting
experimental results.
Select PrimePCR and start a run with a single clickBegin analyzing data in a single step by importing target, reference, and control
information from a PrimePCR run file directly into the plate layout■ Combine data from multiple plates into a Gene Study to rapidly screen large
numbers of targets or samples■ 
Use powerful data visualization tools, such as hierarchical clustering andcolor-matched expression levels, to identify individual targets or clusters
to consider for further investigation■ View data in the PrimePCR Controls Analysis Tool and Reference Gene Selection Tool
in CFX Maestro Software to easily view results of controls and identify ideal reference
genes for your experiment1

Start run2

Apply run file■■

PrimePCR Runs from Start to Finish

PrimePCR
run file3

The run file autopopulates theplate layout with gene names,
making multiplate data analysis
quick and simple.

Automated data analysis

Start runs quickly by choosing PrimePCR in the Startup Wizard to select thevalidated PrimePCR run protocol, then click Start Run. CFX Maestro Software
is fully integrated with PrimePCR products for a fast, streamlined path from data
generation to data analysis.
PrimePCR Analysis Software
For non-CFX platforms, Bio-Rad offers a PrimePCR data analysis solution. Visit
bio-rad.com/PrimePCRsoftware to download the software. Easily upload Cq values
and quickly generate meaningful information from your gene expression experiment.
Use PrimePCR Analysis Software!
bio-rad.com/PrimePCRsoftware

Amplification Reagents and Plastics

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59

8 PCR RUN ANDDATA ANALYSIS
Bio-Rad real-time PCR systems are designed
to deliver outstanding thermal performanceand sensitive optical detection. By pairing provenreal-time instruments with quality PCR reagentsand powerful analysis software, you canconsistently obtain high-quality data to fuelyour research discoveries.
■

■

■

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Amplification Reagents and Plastics

 FX real-time PCR systems efficiently provideCaccurate, reliable data
CFX Maestro Software enables the collection 		and in-depth analysis of real-time data
PrimePCR Analysis Software is a stand-alone 		data analysis tool for non–Bio-Rad instruments

PCR RUN AND DATA ANALYSIS

Overview of CFX Systems and Software

8

Overview of CFX Systems and Software
CFX Real-Time PCR Systems

CFX Maestro Software

Bio-Rad real-time PCR systems are powerful, flexible, and accurate instruments forboth singleplex and multiplex detection of fluorophores. The systems feature thermal
gradient functionality and automation capabilities.

CFX Maestro Software for CFX real-time PCR systems is easy-to-use. This flexible
and powerful software performs data collection, data analysis, and graphing ofreal-time PCR data. You can view and analyze the most relevant data in one screen.

CFX Opus 96 Real-Time PCR System
CFX Opus 384 Real-Time PCR System■ CFX96 Touch Deep Well Real-Time PCR System■ CFX Connect Real-Time PCR System

With CFX Maestro Software you can:

■
■

■

■

■

■

■

Perform automatic statistical analysis in seconds — with just a few mouseclicks you can perform t-tests or analyze your data with 1-way analysis ofvariance (ANOVA)
Extract more meaningful information from your run — analyze data using
bar chart, box and whisker plot, dot plot, clustergram, scatter plot, or volcano plot
Create and export publication-ready graphics — annotate graphs with
P values, text, and arrows to call out specific data. Change colors, fonts, andlegends. Export graphs at any size or resolution for presentations, posters,
or for publication
Easily integrate PrimePCR Assays — use PrimePCR Primers and Plates tosave time on primer design with predesigned and validated primers. Post-run, use
the PrimePCR Controls Analysis Tool to ensure run quality from integrated controls
Work anywhere, on a PC or Mac — with both PC and Mac versions of CFX
Maestro Software, you can analyze your data on your own computer, anytime,
without the need for an Internet connection (Mac version is for data analysis onlyand does not provide instrument control)

Designed for compatibility and integration. CFX Opus Real-Time PCR Systems are automationand integration ready. CFX Maestro Software has an application programming interface that allows for
integration with laboratory information management systems and configuration into other custom
software solutions.

For more information about CFX
systems, download bulletins 5990,
6105, and 7279.

For more information about CFX
Maestro Software, download
bulletin 6900.

Amplification Reagents and Plastics

bio-rad.com

61

Reference
Bustin SA et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55, 611–622.

Visit bio-rad.com/AmpConsumables for more information.
BIO-RAD, DROPLET DIGITAL PCR, HARD-SHELL, and MICROSEAL are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of theirrespective owner.
EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents containing EvaGreen Dye for use in real-time PCR, for research purposes only.
SYBR is a trademark of Thermo Fisher Scientific Inc. Bio-Rad Laboratories, Inc. is licensed by Thermo Fisher Scientific Inc. to sell reagents containing SYBR Green I for use in real-time PCR, for research
purposes only.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the "Products") from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc. Standard Terms and Conditions of Sale,
which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for you to distribute or resell
the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for the development or commercialization of diagnostic assays for use with the Products
without a license from Bio-Rad. The Products and/or their use are covered by U.S.​and foreign patents and/or pending patent applications owned by or under license to Bio-Rad Laboratories, Inc.
See https://www.bio-rad.com/en-us/trademarks.

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