+--------+-------------------------+----------+------------------------+
| su     |                         |          |                        |
| bject: |                         |          |                        |
+--------+-------------------------+----------+------------------------+
| **MAQC |                         |          |                        |
| Study: |                         |          |                        |
| Data   |                         |          |                        |
| Gene   |                         |          |                        |
| ration |                         |          |                        |
| Plan** |                         |          |                        |
|        |                         |          |                        |
| **     |                         |          |                        |
| Dualch |                         |          |                        |
| ip^TM^ |                         |          |                        |
| from   |                         |          |                        |
| Eppe   |                         |          |                        |
| ndorf: |                         |          |                        |
| Gene   |                         |          |                        |
| Expr   |                         |          |                        |
| ession |                         |          |                        |
| Plate  |                         |          |                        |
| form** |                         |          |                        |
+--------+-------------------------+----------+------------------------+
| to:    |                         | only for |                        |
|        |                         | info     |                        |
|        |                         | rmation: |                        |
+--------+-------------------------+----------+------------------------+
|        | MAQC consortium         | EAT      | Christophe Van Huffel  |
|        |                         |          |                        |
|        |                         |          | José Remacle           |
+--------+-------------------------+----------+------------------------+

**[Test Sites]{.underline}**

1\) Test Site : Eppendorf Array Technologies

Contact : Francoise de Longueville

Address : 20, Rue du Séminaire, 5000 Namur, Belgium

Phone : 0032 (81) 725615

2\) Test Site : Cold Spring Harbor

Contact : Eli Hatchwell

Address : Genome research Center, 500 Sunnyside blvd, 11797 Woodbury

Phone : 001 (516)4224121

[]{.mark}

3\) Test Site : MD Anderson

Contact : Lajos Pusztai

> Address : Department of breast medical oncology, PO BOX 301439 /
> HOUSTON, Texas 77230-1439

Phone : 001 (713)792-2817

**[Materials required]{.underline}**

***Eppendorf Products***

-- **Dualchip^TM^ kit** **(3 kits will be provided per site)**

-- **cMaster® RTplus Labeling kit**, Eppendorf (25 reactions) **(2 kits
will be provided)**

-- **Biotin-11-dCTP**, 5 mM (Perkin Elmer, customized) **(2 tubes will
be provided)**

-- **Biotin-11-dATP**, 5 mM (Perkin Elmer, customized) **(2 tubes will
be provided)**

-- **Molecular Biology Grade Water**, (e.g. Eppendorf) **(3 bottle will
be provided)**

-- **Cy3-conjugated IgG Anti biotin**, Jackson Immuno Research
laboratories,

Inc (#200-162-096) **(2 tubes will be provided)**

-- **Tween 20**, e.g. Sigma (# P1379) **(Not provided by Eppendorf)**

--**Thermomixer hybridization equipment** (Eppendorf Thermomixer comfort

and Thermoblock for slides DC) (**3 equipments will be provided)**

-- **Eppendorf CombiSlide Adapter** **(Not provided by Eppendorf)**

-- **DualChip evaluation software** (Eppendorf) (**1 CD will be
provided)**

***Non-Eppendorf Products***

-- Total RNA, Brain (Ambion)(**AB**) -- 100 µg

-- Total RNA, Universal Human Reference RNA (Stratagene) (**SUHRR**) -
100 µg

**[User Manuals]{.underline}**

The manual for the Dualchip^TM^ gene expression plateform will be
provided for each test site. The manual of Dualchip^TM^ kit (version
2.0) is in annexe 1.

**[Experimental Design]{.underline}**

Four RNA samples (2 references RNAs and 2 mixtures)

  ----------------- -----------------------------------------------------
  **RNA**           **Description**

  A                 Stratagene UHRR (SUHRR)

  B                 Ambion Brain Reference RNA

  C                 25% Brain / 75% SUHRR

  D                 75% Brain / 25% SUHRR
  ----------------- -----------------------------------------------------

Five replicates hybridization per sample with independent labelling
reactions will be conducted for the MAQC main study. This experimental
design will be conducted on each test site.

**[Total RNA Preparation and Analysis]{.underline}**

SUHRR will be provided by Stratagene in a solution of 70% ethanol and
0.1 M sodium acetate.

This should be prepared as per the procedure recommended in Stratagene's
Catalog #740000.

AB will be provided in aqueous solution ready for use.

***RNA quantitation and purity assessment:*** RNA should be quantitated
using a NanoDrop ND-100 UV-VIS or equivalent. A260 and A280 should be
recorded for reporting to the MAQC study (Appendix 3:
"MACQ_RNA_Quality_report_template.xls").

***RNA intactness assessment:*** 200 ng quantities of SUHRR and AB
should be run on the Bioanalyzer 2100, three replicates each.
Bioanalyzer traces should be saved, and rRNA Ratio\[28S/18S\] and RIN
values recorded, for reporting to the MAQC study (Appendix 3:
"MACQ_RNA_Quality_report_template.xls").

**[Sample labelling]{.underline}**

Five replicate labeling reactions should be set up for each sample as
follows:

-- Procedure described in Eppendorf Dualchip kit manual (point
8.3.2.1.A, page 19) should be followed (Annexe 1).

-- 10 µg of total RNA should be input per reaction.

-- All 20 target preparations (4 RNAs X 5 replicates) should be
[processed together]{.underline} starting on the same day by a single
person to minimize run-to-run variability.

**[Sample Hybridization]{.underline}**

Five replicate hybridization per sample should be performed.

-- Procedure described in Eppendorf Dualchip kit manual (point
8.3.2.1.A, page 21) should be followed (Annexe 1).

-- All 20 hybridizations (20 arrays, 10 slides (2 array per slides))
should be [processed together]{.underline} starting on the same day by a
single person to minimize run-to-run variability (in one batch).

-- During all processing steps, the order of the 20 microarrays being
used should be randomized, i.e., Hybridization Solutions should be
hybridized to randomly selected microarrays, scan order should be
randomized.

-- Microarrays should be hybridized at 60°C for 16 hours.

-- Microarrays should be washed in one batch of 10 slides .

-- Microarrays should be washed with Eppendorf Washing Buffer 1 as
described in Eppendorf Dualchip kit manual (point 8.3.2.1.C, page 26)
(Annexe 1).

-- Slides should be scanned as soon as possible after washing.

**[Data Acquisition]{.underline}**

***Microarray scanning:*** All 20 microarrays should be scanned
following the procedure described in Eppendorf Dualchip kit manual
(point 8.4.1., page 41) (Annexe 1).

***Data extraction:*** Data should be extracted using Dualchip
Evaluation software, version 1.0 using the procedure described in
Eppendorf Dualchip kit manual (point 8.4.2. A. B., page 41) (Annexe 1).