![](media/image1.png)

***StaRT*-PCR^TM^ Platform Data Generation Plan**

**AND**

**Standard Operating Procedures for MAQC Study**

**Test Site**

> Test site: Gene Express, Inc.

Contact Person: James C. Willey, M.D.

Address: 975 Research Drive

Toledo, OH 43614-2788

Phone: (419) 383-3541

E-mail: <jwilley@umedohio.edu>

Contact Person: Elizabeth Herness Peters, Ph.D.

Address: 975 Research Drive

Toledo, OH 43614-2788

Phone: (419) 380-9930

E-mail: [ehpeters@geneexpressinc.com](mailto:jwilley@umedohio.edu)

## Introduction

> The purpose of this document is to describe the procedures that will
> be preformed using the *StaRT*-PCR^TM^ platform for the MAQC study.

## Materials

Gene Express, Inc. Products

+-----------------------------------------------------------------------+
| ### 1. Standardized Mixtures of Internal Standards^TM^ ( SMIS^TM^)    |
+-----------------------------------------------------------------------+
| 2\. Gene-Specific Primers (forward + reverse at a concentration of    |
| 0.05 µg/ µl each)                                                     |
+-----------------------------------------------------------------------+
| 3\. HT S-GEM Suite^TM^ software program                               |
+-----------------------------------------------------------------------+

#### RNA Samples

+-----------------------------------------------------------------------+
| ### 1. S                                                              |
| tratagene Universal Human Reference RNA (UHRR) -- Catalog No. 740000  |
+-----------------------------------------------------------------------+
| 2\. Ambion Human Brain Reference RNA (AB) -- Catalog No. *TBA*        |
+-----------------------------------------------------------------------+
| 3\. RNA mixture of 25% AB vs. 75% UHRR                                |
+-----------------------------------------------------------------------+
| 4\. RNA mixture of 75% AB vs. 25% UHRR                                |
+-----------------------------------------------------------------------+

> Disposables

+-----------------------------------------------------------------------+
| ### 1. Disposables are listed in the SOP manual an                    |
| d include items such as clean Kim wipes, microcentrifuge tubes, pipet |
| te tips, 96 well plates, buffer troughs, plastic adhesive seals, etc. |
+-----------------------------------------------------------------------+

> User Manual

+-----------------------------------------------------------------------+
| #                                                                     |
| ## 1. Standardized Expression Measurement Center (SEM^TM^) SOP manual |
+-----------------------------------------------------------------------+

######  Reagents

+-----------------------------------+-----------------------------------+
| ### ITEM                          | ### SOURCE                        |
+-----------------------------------+-----------------------------------+
| First Strand cDNA buffer          | Invitrogen Cat. No. Y00146        |
+-----------------------------------+-----------------------------------+
| dNTPs, 10 mM                      | Promega Cat. No. C1141            |
+-----------------------------------+-----------------------------------+
| Oligo dT Primer, 500 g/l          | Promega Cat. No. 15199905         |
+-----------------------------------+-----------------------------------+
| RNasin, 40 units/l                | Promega Cat. No. 15140508         |
+-----------------------------------+-----------------------------------+
| MMLV Reverse Transcriptase, 200   | Invitrogen Cat. No. 28025-013     |
| units/ml                          |                                   |
+-----------------------------------+-----------------------------------+
| Sterile RNase/DNase free water    | Invitrogen Cat. No. 10977015      |
+-----------------------------------+-----------------------------------+
| Tris-EDTA Buffer, pH 8.0 and pH   | Fisher Cat. No. BP1338-1          |
| 7.4                               |                                   |
+-----------------------------------+-----------------------------------+
| 10X PCR Buffer without Mg^2++^    | Invitrogen Cat. No. Y02028        |
+-----------------------------------+-----------------------------------+
| MgCl~2~, 50 mM                    | Invitrogen Cat. No. Y02016        |
+-----------------------------------+-----------------------------------+
| Platinum Taq polymerase, 5U//l    | Invitrogen Cat. No. 10966-026     |
+-----------------------------------+-----------------------------------+
| DNA Buffer Solution               | Invitrogen Cat. No. Y02028        |
+-----------------------------------+-----------------------------------+
| DNA Ladder                        | Caliper Life Sciences Cat. No.    |
|                                   | P/N 760124                        |
+-----------------------------------+-----------------------------------+
| DNA Gel-Dye Mixture               | Caliper Life Sciences Cat. No.    |
|                                   | P/N 760124                        |
+-----------------------------------+-----------------------------------+
| HT DNA 5000SE 30 Marker           | Caliper Life Sciences Cat. No.    |
|                                   | P/N 760124                        |
+-----------------------------------+-----------------------------------+

> Equipment

+-----------------------------------+-----------------------------------+
| ### ITEM                          | ### SOURCE                        |
+-----------------------------------+-----------------------------------+
| Block Thermocycler                | MWG                               |
+-----------------------------------+-----------------------------------+
| Agilent 2100 Bioanalyzer          | Agilent Technologies              |
+-----------------------------------+-----------------------------------+
| MultiPROBE II HT EX Liquid        | Perkin Elmer                      |
| Handler                           |                                   |
+-----------------------------------+-----------------------------------+
| Caliper AMS90 SE                  | Caliper                           |
+-----------------------------------+-----------------------------------+
| AMS-90 Sipper Chip                | Caliper                           |
+-----------------------------------+-----------------------------------+
| Centrifuge                        | Beckman                           |
+-----------------------------------+-----------------------------------+
| Laminar Flow Hoods                | Baker and Hemco                   |
+-----------------------------------+-----------------------------------+
| Pipettes                          | Rainin and Pipetman               |
+-----------------------------------+-----------------------------------+
| Freezer, -80C                     | Sanyo                             |
+-----------------------------------+-----------------------------------+
| Freezer, -20C                     | LRP                               |
+-----------------------------------+-----------------------------------+
| Refrigerator, 4C                  | Kenmore                           |
+-----------------------------------+-----------------------------------+

##### Experimental Design

The RefSeq numbers of a total of 90 to 100 genes will be matched to the
MAQC 1,042 gene list. The remaining genes, up to a total of 200 genes,
will be selected from the list of available *StaRT*-PCR^TM^ gene assays,
located at the following web address
<http://www.geneexpressinc.com/products2.asp>, and approved by the MAQC.

Three replicate measurements for each gene will be obtained for each of
the four mRNA samples listed above. The loading control genes will be
ACTB and GAPD.

***StaRT*-PCR^TM^** Gene Assay Workflow

+-------------+-------------+-------------+-------------+-------------+
| ### Day 1   | ### Day 2   | ### Day 3   | ### Day 4   | ### Day 5   |
+-------------+-------------+-------------+-------------+-------------+
| Receive     | RNA         | E screen    | C Screen    | Replicate   |
| Sample      | Evaluation  |             |             | TA          |
|             |             |             |             | M           |
|             |             |             |             | easurements |
+-------------+-------------+-------------+-------------+-------------+
| Login       | Reverse     |             |             |             |
| Sample      | Tr          |             |             |             |
|             | anscription |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Store       | Calibration |             |             |             |
| Sample      |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+

+-------------+-------------+-------------+-------------+-------------+
| ### Day 6   | ### Day 7   | ### Day 8   | ### Day 9   | ### Day 10  |
+-------------+-------------+-------------+-------------+-------------+
| Replicate   | Replicate   | Replicate   | Replicate   | Replicate   |
| TA          | TA          | TA          | TA          | TA          |
| M           | M           | M           | M           | M           |
| easurements | easurements | easurements | easurements | easurements |
+-------------+-------------+-------------+-------------+-------------+

+-------------+-------------+-------------+-------------+-------------+
| ### Day 11  | ### Day 12  | ### Day 13  | ###         | ###         |
+-------------+-------------+-------------+-------------+-------------+
| Replicate   | Replicate   | Prepare     |             |             |
| TA          | TA          | Final       |             |             |
| M           | M           | Report      |             |             |
| easurements | easurements |             |             |             |
+-------------+-------------+-------------+-------------+-------------+

1.  **Sample Receipt, Login, and Initial Storage of Samples**

> All personnel will follow SOP0001 for receipt, login, and initial
> storage for the four (4) mRNA samples. Samples will be stored at -80C.

1.  New Order Function will be used to begin an order by following
    SOP0035.

2.  A SOURCEPLATE.csv file will be prepared by following SOP0017.

3.  Prepare SOURCEPLATE(s).

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2.  **RNA Preparation and Analysis**

> Four (4) mRNA samples, designated as Stratagene Universal Human
> Reference RNA, Ambion Brain Reference RNA, and two titration mixtures
> will be received from Ambion at a concentration 1 mg/ml per tube in a
> total volume of 50 l.
>
> 2.1 RNA quantity assessment will be preformed on each sample using the
> Agilent Bioanalyzer and recording the 28S/18S ratio and RIN, if
> available.
>
> All personnel will follow SOP0002 for RNA quantity assessment.

2.2 RNA quality assessment will be performed using 200 ng of each sample
and assessed with the Agilent Bioanalyzer. All personnel will follow
SOP0002 for RNA quality assessment.

3.  **Reverse-Transcription of RNA to cDNA**

> All personnel will follow SOP0004 for reverse transcription of mRNA
> samples to cDNA using oligo dT priming and MMLV reverse transcriptase.

4.  **Quantitative Calibration**

> Each of the cDNA samples will be Quantitatively Calibrated according
> to the reference gene (ACTB) such that 1 l of cDNA sample when
> included into the PCR reaction with 600,000 -actin molecules yields
> both native and internal standard PCR products that are approximately
> equal in peak height and area under the curve according to the SOPs of
> Gene Express, Inc. and the HT S-GEM Suite^TM^ software program.

1.  Each cDNA sample will be serially 10-fold diluted using the
    MultiPROBE II HT EX Liquid Handler according to SOP0052.

2.  cDNA quantitative calibration reactions will be prepared in
    microtiter plates by the MultiPROBE II HT EX Liquid Handler using
    SOP0015 and completed using SOP0005.

3.  PCR reactions will be prepared in microtiter plates by the
    MultiPROBE II HT EX Liquid Handler following SOP0018, SOP0023,
    SOP0024 and SOP0025.

4.  PCR amplification of samples prepared in 4.3 will be performed using
    a MWG Block Thermocycler and following SOP0013.

5.  After amplification is complete, a LabChip 90 sipper chip will be
    prepared using SOP0006.

6.  The PCR reaction products from 4.4 will be electrophoretically
    separated and quantified with the Caliper AMS90 SE by using SOP0028.

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5.  Data from the Caliper AMS90 SE will be exported and a report file
    will be generated using SOP0009.

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5.  **E Screen**

####### Each gene will be assessed with E level SMIS^TM^ containing 600 molecules of internal standards. The E screen results determine which level of SMIS^TM^ is required during High Throughput PCR.

1.  PCR reactions will be prepared using 1 l of calibrated cDNA and 1 l
    of E SMIS^TM^ following SOP0018, SOP0023, SOP0024 and SOP0025.

2.  PCR reactions will be performed using a MWG Block Thermocycler and
    following SOP0013.

3.  After amplification is complete, a LabChip 90 sipper chip will be
    prepared using SOP006.

4.  The PCR reaction products from 5.2 will be electrophoretically
    separated and quantified with the Caliper AMS90 SE by using SOP0028.

> 5.5 Data from the Caliper AMS90 SE will be exported and a report file
> will be generated using SOP0009.

6.  **Transcript Abundance (TA) Measurements**

> *StaRT*-PCR^TM^ will be performed using an Internal Standard
> Competitive Template (CT) within the PCR reaction for transcript
> abundance measurement of each gene. *StaRT*-PCR^TM^ does this through
> inclusion of a Standardized Mixture of Internal Standards ^TM^
> (SMIS^TM^) and gene specific forward and reverse primers in transcript
> abundance measurement of each gene. Based on results from Quantitative
> Calibration and E screen, 1 l of calibrated cDNA sample, and 1 l of
> the appropriate SMIS^TM^ (A, B, C, D, E, or F) will be PCR amplified.
> Proper selection of SMIS ^TM^ A thru F enables measurement of target
> gene expressed across a more than 7 log~10~ range. For target genes in
> balance with 1 l of A, B, C, or D SMIS^TM^, 10-fold dilution of both
> cDNA and SMIS ^TM^ will be used. Thus, cDNA and SMIS ^TM^ containing
> approximately 60,000 molecules of b-actin transcript and internal
> standard, respectively, will be in each PCR reaction.

1.  PCR reactions will be prepared following SOP0018, SOP0023, SOP0024
    and SOP0025.

2.  PCR reactions will be performed using a MWG Block Thermocycler and
    following SOP0013.

3.  After amplification is complete, a LabChip 90 sipper chip will be
    prepared using SOP006.

4.  The PCR reaction products from 6.2 will be electrophoretically
    separated and quantified with the plate with the Caliper AMS90 SE by
    using SOP0028.

> 6.5 Export and generate a report file using SOP0009.

7.  **Data Acquisition**

> *StaRT*-PCR^TM^ transcript abundance measurements will be obtained
> using the Caliper AMS90^TM^ data by analyzing the area under the
> curve. Data will be registered and automatically analyzed using the HT
> S-GEM Suite^TM^ software program. The data will be exported into a
> text file.

8.  **Data Management and Quality Control Specifications**

> *StaRT*-PCR^TM^ Data are automatically processed and analyzed as
> described in SOP0009. Quality control specifications include
> appropriate negative and positive control wells on each 96 well plate.
> Peaks must meet a minimum peak height and peak area requirement. Both
> Native and Internal Standard peak base pair sizes must lie within a
> maximum designated range of ± 35 base pairs.

9.  **Final Report**

The final data report will contain the average of the three replicate TA
measurements, the standard deviation and the coefficient of variation.
The final report will be sent directly to the MAQC via electronic file.