**MAQC Main Study Plan (Draft)**

***Applied BioSystems Gene Expression Arrays and Assays***

**Part I: Microarray Study**

A.  **Test sites:**

    1.  Test site I: Applied BioSystems R&D\
        Contact Person: Lu Zhang\
        Address: 850 Lincoln Centre Drive, MS447, Foster City, CA 94404\
        Phone: 650-638-5045

    2.  Test site II: EPA/NHEERL\
        Contact Person: David Dix\
        Address: Reproductive Toxicology Division (MD-72), Nat\'l\
        Health&Environmental Effects Research Lab/Office of R&D, U.S.\
        Environmental Protection Agency, Research Triangle Park, NC\
        27711\
        Phone: 919-541-2701

    3.  Test site III: Vanderbilt University\
        Contact Person: Shawn Levy\
        Address: Vanderbilt Microarray Shared Resource, Vanderbilt
        University, 465\
        21^st^ Ave South, MRBIII Room 9274, Nashville, TN 37232

> Phone: 615-936-3003

B.  **Materials and Equipment Required:**

-   ***RT-IVT labeling:***

1.  NanoAmp^TM^ RT-IVT labeling kit (P/N 4365715) **(Provided by ABI)**

2.  NanoAmp^TM^ Rt-IVT labeling kit protocol (P/N 4365710)

3.  Quick Reference Card, NanoAmp^TM^ RT-IVT labeling kit (P/N 4365711)
    **(Provided by ABI)**

4.  96-Well GeneAmp PCR System 9700 (P/N4314878) or other equivalent PCR
    machine

> All other reagents and equipments required for RT-IVT labeling are
> described in NanoAmp^TM^ Rt-IVT labeling kit protocol (P/N 4365710)
> Materials and Equipment section (Page 3-7)

-   ***Microarray hybridization and Chemiluminescent detection***:

5.  Chemiluminescent detection Kit (P/N 4342142) **(Provided by ABI)**

6.  Protocol, Chemiluminescent detection Kit (P/N 4339627)

7.  Quick Reference Card, Chemiluminescent detection Kit (P/N 4346875)
    **(Provided by ABI)**

8.  Applied BioSystems Microarray Wash Tray (P/N 4352927)

9.  Applied Biosystems 1700 Chemiluminescent Microarray Analyzer (P/N
    4338036)

10. Expression Array System Analyzer Software Version 1.1.1

11. Human Genome Survey Microarray Version 2.0 (12 pack, P/N 4359031)
    **(Provided by ABI)**

12. Anti-Digoxigenin-AP, Fab fragments: 150 U (200 ul) (By Roche Applied
    Science, P/N 1093274) **(Provided by ABI)**

13. Digoxigenin-UTP:200nmol (57 ul) (By Roche Applied Science,
    P/N 3359247) **(Provided by ABI)**

14. Chemiluminescent Microarray Analyzer User Guide (P/N 4338852)

15. Chemiluminescent Microarray Analyzer Chemistry Guide (PN 4338853)

16. Hybridization Oven at 55^0^C (Recommended: Signature Benchtop
    Shaking Incubator, Model 1575 by VWR Scientific, or Infors HT
    Minitron Incubator Shaker)

17. VWR Rocking Platform Shaker, Single-Tier Platform (VWR, P/N
    40000-300 for North America, P/N 40000-302 for Europe and other
    regions)

> All other reagents and equipments required for microarray
> hybridization and chemiluminescent detection are described in
> Chemiluminescent detection Kit protocol (P/N 4339627) Materials and
> Equipment section (Page 3-7)

-   ***Other Materials:***

> Four Total RNAs with each greater than 15 ug (Provided by Ambion and
> Stratagene): Universal Human Reference RNA (Stratagene, SUHRR)
> (**A**), Brain (Ambion) (**B)**, 25% Brain / 75 % SUHRR (**C**), 75%
> Brain / 25 % SUHRR (**D**)
>
> Agilent Bioanalyzer 2100 and RNA 6000 Nano kit

C.  **Experimental Design:**

> Five technical replicates, which include labeling and corresponding
> microarray hybridization reactions, will be processed for all four
> total RNA samples (A, B, C and D respectively). One individual will
> label all samples on one day. The microarray hybridizations will be
> split into two groups run on two separate days. The same person should
> hybridize microarrays on the two separate days. On day 1, three
> replicates for all four samples (A, B, C and D) will be hybridized and
> detected. On day 2, the remaining two replicates for all four samples
> (A, B, C and D) will be hybridized and detected. The hybridization day
> for each array will be documented and submitted to the MAQC along with
> the primary data.

D.  **Experimental Procedures:**

    1.  Total RNA QC:

> Upon receiving, all four total RNA samples (A, B, C and D) will be
> quantified with a spectrophotometer. The concentration and OD260/OD280
> ratio for each sample will be documented in the template provided by
> MAQC project (MAQC_RNA_Quality_Report_Template.xls).
>
> RNA integrity will be checked by running 250ng total RNA sample on
> Bioanalyzer system. 28S/18S ratio and RIN numbers will be documented
> in the same template as above.
>
> Note: If a RIN number \< 9.0 is observed, report the finding to MAQC
> team and do not proceed further.

2.  Total RNA labeling:

> **1 ug** of each total RNA sample will be used as the starting
> material and the labeling reaction will be performed according to the
> procedure described in the Applied BioSystems NanoAmp^TM^ Rt-IVT
> labeling kit protocol (P/N 4365710) and Quick Reference Card,
> NanoAmp^TM^ RT-IVT labeling kit (P/N 4365711). All 24 samples will be
> labeled at the same process by same person.

3.  Labeled cRNA QC:

> All the labeled cRNA will be quantified using the spectrophotometer
> and cRNA quality will be checked using Bioanalyzer system with 250ng
> samples. The cRNA concentration, cRNA size distribution and a median
> size value will be reported in the template provided by MAQC project
> (MAQC_RNA_Quality_Report_Template.xls).

4.  Microarray Hybridization:

> The hybridization reaction will be performed according to the
> procedure described in the Protocol, Chemiluminescent detection Kit
> protocol (P/N 4339627) and Chemiluminescent detection Kit Quick
> Reference Card (P/N 4346875)

5.  Chemiluminescent detection:

> The Chemiluminescent detection will be performed according to the
> procedure described in the Protocol, Chemiluminescent detection Kit
> protocol (P/N 4339627) and Chemiluminescent detection Kit Quick
> Reference Card (P/N 4346875) with the following modifications (Quick
> Reference Card has been referred here).
>
> At the Performing Hybridization Washes step, these changes should be
> followed:
>
> **3. Perform hybridization wash 1:**
>
> **a. Cover the wash tray with the wash tray cover, then agitate on the
> rocking platform shaker for 10 minutes, instead of 5 minutes**

**4. Perform hybridization wash 2:**

> **a. Cover the wash tray with the wash tray cover, then agitate on the
> rocking platform shaker for 10 minutes, instead of 5 minutes**

6.  Data Acquisition:

> After the microarray has been processed, the images will be generated
> according to the guidance described in the Protocol, Chemiluminescent
> detection Kit protocol (P/N 4339627) and Chemiluminescent detection
> Kit Quick Reference Card (P/N 4346875). The samples should following
> the naming convention guide provided by MAQC documents
> (MAQC_RNA_Quality_Report_Template.xls). The output data will be
> generated using the Expression Array System Analyzer Software Version
> 1.1.1 and both microarray images (.tif) and output data will be
> delivered to the MAQC.

7.  Data Analysis Guide:

> The initial data analysis could follow the guidance in the following
> documents: Chemiluminescent Microarray Analyzer User Guide (P/N
> 4338852) and Chemiluminescent Microarray Analyzer Chemistry Guide (PN
> 4338853). Advanced data analysis guide will be provided separately.

**Part II: TaqMan RT-PCR validation**

A.  **Test sites**

    1.  Test site I: Applied BioSystems R&D\
        Contact Person: Lu Zhang\
        Address: 850 Lincoln Centre Drive, MS447, Foster City, CA 94404\
        Phone: 650-638-5045

B.  **Materials Required**

***Applied Biosystems Products*:**

# TaqMan Gene Expression Assays (P/N 43311882 ) -1000 Inventoried Assays

# Applied Biosystems 7900HT Fast Real-Time PCR System

-   SDS Software v2.2.1 or higher for the 7900HT System (P/N4352620)

-   \*RNAse Inhibitor (P/N 8080119)

-   \*High-Capacity cDNA Archive Kit (P/N 4322171)

-   \*TaqMan® Universal PCR Master Mix (2X), No AmpErase® UNG (P/N
    4352042)

-   \*ABI Prism® 96-Well Optical Reaction Plate with Barcode (code 128)
    (P/N 4306737)

-   \*ABI Prism® 384-Well Optical Reaction Plate with Barcode (code 128)
    (P/N 4309849)

-   \*ABI Prism® Optical Adhesive Covers (P/N 4311971)

-   TaqMan Low Density Array Endogenous Control Panel (P/N 4367563)

-   7900HT Low Density Array Upgrade (P/N 4329012)

-   TaqMan® Gene Expression Assays Protocol (P/N 4333458)

## High-Capacity cDNA Archive Kit Protocol (P/N 4322169)

-   Applied Biosystems 7900HT Fast Real-Time PCR System and SDS
    Enterprise Database User Guide (P/N 4347828)

-   TaqMan Low Density Array User Guide (P/N 4319399)

-   \*Reagents and consumables provided for \> 4000 TaqMan Assays

***Non-Applied Biosystems Products:***

-   Total RNA, Brain (Ambion) - 150 µg

-   Total RNA Universal Human Reference RNA (UHR) (Stratagene) - 150µg

-   Total RNA, Mixture 25% Brain, 75% UHR -- 150µg

-   Total RNA, Mixture 75% Brain, 25% UHR -- 150µg

# All other non-Applied Biosystems reagents and equipment required are described in Applied Biosystems Documents TaqMan® Gene Expression Assays Protocol (P/N 4333458), High-Capacity cDNA Archive Kit Protocol (P/N 4322169), TaqMan Low Density Array User Guide (P/N 4319399)

C.  **Experimental Design**

1000 genes will be selected by MAQC for QRT-PCR analysis with TaqMan®
Gene Expression Assays from a list of \>5000 RefSeqs that showed perfect
match to a Taqman® Assay and to the probe sequences on the seven
microarray platforms (Affymetrix, Agilent, AB Microarray, Combimatrix,
GE Healthcare, Illumina, and NCI_Operon)

. The final selection will be based on expression data generated in
Pilot Study 1 and will include 100 genes which showed the most
variability and 100 genes with the best sample specificity (50 brain, 50
UHR) across platforms from Pilot Study 1. \~175 genes with RefSeqs that
match Eppendorf and Genospectra probe sets are also included. The
remainder will include a random selection of genes that cover whole
intensity and log ratio ranges.

TaqMan® Gene Expression Assays will be done in four replicates for each
RNA sample. 10ng total cDNA (converted from total RNA) in 10μl final
volume will be used for each replicate assay. 4 selected endogenous
controls to use for normalization will be run on each 384 reaction
plate: 18S(Hs99999901_s1), UBC(Hs00824723_m1), HPRT(Hs99999909_m1),
POLR2A(Hs00172187_m1).

### Total RNA Preparation and QC process

-   The RNA samples used for microarray analysis will also be used for
    TaqMan® Assay. Please refer to previous section on Microarray Study
    for RNA preparation and QC process.

D.  **cDNA preparation and QC process**

Synthesis of cDNA from total RNA samples is the first step in TaqMan
Gene Expression Assay.

### Synthesis will be carried out according to High-Capacity cDNA Archive Kit Protocol (P/N 4322169) .

### 10μg total RNA per 100μl reactions for each sample (10 reactions per sample).

### All reactions from the same RNA sample will be pooled.

### cDNA will be tested by running each pooled sample on a TaqMan® Low Density Endogenous Control Panel. Refer to TaqMan® Low Density Array User Guide for detailed protocol.. 

> 100ng cDNA is made up to 50μl in TE final volume. The sample is then
> combined with 50μl TaqMan® Universal PCR Master Mix (2X), No AmpErase®
> UNG (P/N 4352042). Final concentration is 100ng cDNA /100ul 1X Master
> Mix.
>
> 100μl of each sample is then added into a sample port on the low
> density array.
>
> The array is centrifuged, sealed, and run on 7900HT as described in
> User Guide.
>
> Data is analyzed with SDS 2.1 or higher software.
>
> C~T~ values will be compared to results generated from the Pilot Study
> II_Tissue Titration Study.

E.  **TaqMan Gene Expression Assay**

# The procedure described in TaqMan® Gene Expression Assays Protocol (P/N 4333458) will be followed with the following modifications:

-   Use 10 ng cDNA per reaction.

-   The final volume is 10μl for each reaction.

-   Reaction mixture for 4 reactions (plus additional volume for
    overage) will be prepared in a 96-well plate and replicate assays
    transferred to 384-well Optical Reaction Plate using robotic methods
    (Biomek FX).

-   Reaction Plate will be run on 7900HT System using Standard Thermal
    Cycler mode, Sample Volume set at 10μl.

```{=html}
<!-- -->
```
-   Universal Cycling conditions: 10' at 95ºC; 15" at 95ºC, 1' 60ºC, 40
    cycles.

### Data Acquisition and Analysis

Data will be generated and output using SDS 2.2 or higher software in
text format. Data will be analyzed using ΔCt (absolute Ct value --
endogenous control Ct value) and ΔΔ C~T~ (Δ C~T~ for each gene -- Δ C~T~
for a common reference gene) methods. The endogenous control with the
least variation across all the samples and plates within one sample will
be used for normalization to calculate Δ C~T~ t for each individual
gene.

###  {#section .unnumbered}

Our brief guidelines for validation of microarray results using TaqMan®
Assay data as standard are in three categories:

1.  Validate detection sensitivity and accuracy from each microarray
    platform:

2.  Fold change correlation with TaqMan® data (ΔΔ C~T~ method) for all
    microarray platform

3.  Differential expression sensitivities and accuracy by each
    microarray platform

The genes could be evaluated as a whole set (1041 total) or divided into
subcategories according to the original design:

> Subcategory 1: Total randomized genes (Number to be finalized)
>
> Subcategory 2: Sample specific genes (110)
>
> Subcategory 3: Most variable genes (100)

More details will be available in the upcoming Data Analysis guide.