**MAQC Study -- Data Generation Plan** (*DRAFT)*

***Genospectra QuantiGene Platform***

**Test Site**

1.  Genospectra, Inc.

> Contact person: Yuling Luo
>
> Address: 6519 Dumbarton Circle, Fremont, CA 94555
>
> Phone: 510.818.2623
>
> Email: yluo@genospectra.com

**Materials Required**

***Genospectra Products***

1.  • QuantiGene kits

    • Gene-specific probe sets for over 200 genes

***Non-Genospectra Products***

1.  • Total RNA, Brain (Ambion)(AB)

    • Total RNA, Universal Human Reference RNA (Stratagene) (UHRR)

    • Total RNA, Mixture of 75% AB + 25% UHRR

    • Total RNA, Mixture of 25% AB + 75% UHRR

***Equipment and consumables***

6.  • General laboratory equipment include a 96-well plate-based
    luminometer (Lmax, Molecular Device) and a hybridization incubator
    (Hybaid) capable of maintaining temperature between 46ºC and 53ºC.

    • General consumables are listed in the QuantiGene assay SOP manual

***User Manuals***

1.  • Genospectra QuantiGene Assay SOP Manual

**Experimental Design**

Minimal of 200 genes will be selected for the QuantiGene assay and
agreed to by MAQC. The 200 gene will be matched within the ABI's 1,000
gene list, which will be matched within the list of 5,000 RefSeq genes
shared by all microarray platforms. The 200 genes will be selected to
cover different expression level, fold changes, and some genes showing
cross-platform discrepancy in the pilot study.

**Agreement need to be reached among Genospectra, ABI, and Stratagene
for the platform validation assays**. Three, four or five replicate
measurements for each gene can be processed for each of the following
mRNA samples, depending on the agreement among Genospectra, ABI, and
Stratagene:

1.  1\. UHRR (100%)

    2\. AB (25%) vs. UHRR (75%)

    3\. AB (75%) vs. UHRR (25%)

    4\. AB (100%)

**Total RNA Preparation and Analysis**

It is assumed that all four total RNA samples will be provided in
aqueous solution ready for use. The total RNA samples will be stored at
-80 ^o^C and will be used directly in the QuantiGene assay, unless there
is a need to dilute the sample further. No additional manipulation is
required for the QuantiGene assay. As a result, we rely on the quality
assessment of the original total RNA samples by Ambion and Stratagene.

***QuantiGene® assay conditions:***

1\. Stock total RNA samples will be diluted to \[40ng/ul\] in RNase Free
water.

> 2\. 100 to 800ng of total RNA will be added to each well of QuantiGene
> assay, depending on the gene expression level.

3\. Hybridization assay will be carried out per QuantiGene® manual.

> 4\. Background signal for each gene will be assessed in a QuantiGene
> assay with no sample RNA added, just the probe set.

**Data Acquisition and Analysis**

QuantiGene assay signal is measured by a luminometer, and the Relative
Luminescent Unit (RLU) data will be directly read from the luminometer
machine. The data will be exported into an Excel file and reported
directly to MAQC.

For those genes (e.g. selected genes from MAQC pilot II study) whose
expression in AB and UHRR is substantially different (e.g. over 100: 1
ratio), we expect an assay signal ratio of 100: 75:25:0 ratio for the
four total RNA samples. The closeness to the expected 100:75:25:0 ratio
will be a measurement of the assay platform accuracy.

Once the assay platform accuracy is established, the relative ratio of
the 200 genes among different samples will be assessed, and their
correlation with the data from other platforms will be compared.

**Overview of the QuantiGene Workflow**

**Day One:**

## 

## Prepare the QuantiGene^®^ Reagents and gene-specific Probe Set

## Prepare RNA samples for the selected genes.

A.  Follow Experimental Layout on Plate Map.

**Day Two:**

D.  Prepare the QuantiGene Reagents used for the target mRNA.

## Amplify the Signal and Detect target mRNA.

E.  Analyze the Data.

# 

**QuantiGene** **Assay SOP**

PURPOSE:

This protocol describes the procedure for performing, single-plex
QuantiGene assays with purified RNA using the LMAX luminometer.

SCOPE:

This procedure applies to all R&D personnel performing a QuantiGene
single-plex assay using purified RNA or IVTs as the sample source.

KEY SPONSOR and responsibility:

The R&D Manager is responsible for the content, updating and
implementation of this document.

eQUIPMENT:

1.  Pipettes, single and multichannel

2.  Vortex mixer

3.  LMAX luminometer

4.  Incubators: 46° +/-1.0°C and 53° C +/- 1.0° C

5.  Quick spin bench top micro-centrifuge

mATERIALS

1.  QuantiGene Assay Kit (at 4°C - capture plates, amplifier, label
    probe, substrate; at 15-30°C - amplifier/label probe diluent, lysis
    mixture)

2.  Plastic pipette tips, aerosol resistant

3.  Disposable reagent reservoirs, 25 and 100 mL capacity

4.  Plate Sealers (Bioexpress, Cat. No. T-3025-8B)

5.  10X or 1X Wash Buffer

6.  10% Lithium Lauryl Sulfate solution

7.  Capture Buffer

8.  1 mg/mL tRNA (Blocking Agent)

9.  Purified total RNA, mRNA, or IVT RNA

10. Appropriate Probe Set(s) with pooled Label Extenders, pooled Capture
    Extenders and pooled Blocking Probes

11. Nuclease Free water

**SOP Procedure:**

**Day One:**

+-----+----------------------------------------------------------------+
| **  | **Action**                                                     |
| Ste |                                                                |
| p** |                                                                |
+-----+----------------------------------------------------------------+
| **  | Bring the **Capture Plate** and target-specific probes to room |
| 1** | temperature.                                                   |
+-----+----------------------------------------------------------------+
| **  | Check to be sure the **Lysis Mixture** does not have           |
| 2** | precipitates. If you observe precipitates, then perform the    |
|     | following:                                                     |
|     |                                                                |
|     | a\. Place the bottle in a 37ºC water bath.                     |
|     |                                                                |
|     | b\. Gently swirl contents occasionally until all precipitates  |
|     | dissolve.                                                      |
+-----+----------------------------------------------------------------+
| **  | Prepare the **Concentrated Probe Sets** for the target RNA of  |
| 3** | interest by adding 50 µl each of pooled 1X CE, (Capture        |
|     | Extender, 50 fmol/µl), 1X LE, (Labeled Extender, 200           |
|     |                                                                |
|     | fmol/µl), and 1X BL, (Blocking Probe, 100 fmol/µl), probes to  |
|     | 850 µl of **Lysis Mixture**                                    |
|     |                                                                |
|     | (from QuantiGene kit).                                         |
|     |                                                                |
|     | **Note: This preparation of concentrated probe set is enough   |
|     | for one 96-well plate.**                                       |
+-----+----------------------------------------------------------------+
| **  | Prepare **Lysis Working Reagent**: Add 15 µl of yeast tRNA, to |
| 4** | 6 ml of **Lysis Mixture**                                      |
+-----+----------------------------------------------------------------+
| **  | Depending upon the expected abundance of the specific mRNA to  |
| 5** | be measured, dilute                                            |
|     |                                                                |
|     | the RNA sample with **Lysis Working Reagent** (see Step 4,     |
|     | above) to a final concentration of 0.02 to 2 µg/10 µl, if      |
|     | using mRNA dilute to a final concentration of 0.4 to 40 ng/10  |
|     | µl.                                                            |
|     |                                                                |
|     | **Note:** For a gene with a moderate level of expression, 0.2  |
|     | µg/10 µl or 4 ng/10 µl of mRNA are recommended.                |
+-----+----------------------------------------------------------------+
| **  | Dispense 50 µl of **Capture Buffer,** (Catalog \#              |
| 6** | QGR-004-002), to the **Capture Plate** well                    |
+-----+----------------------------------------------------------------+
| **  | Dispense 40 µl of **Lysis Mixture** to the **Capture Plate**   |
| 7** | well.                                                          |
+-----+----------------------------------------------------------------+
| **  | Dispense 10 µl of **RNA sample** to the **Capture Plate**      |
| 8** | well.                                                          |
+-----+----------------------------------------------------------------+
| **  | Dispense 10 µl of **Concentrated Probe Sets** to the **Capture |
| 9** | Plate** well.                                                  |
+-----+----------------------------------------------------------------+
| **1 | Seal the **Capture Plate** with a plate sealer. Incubation the |
| 0** | sealed plate at 53°C for 16-20 hours.                          |
+-----+----------------------------------------------------------------+

**Day Two:**

**Procedural Guidelines**

-   We recommend preparing the **Substrate**, **Amplifier** and **Label
    Probe** immediately before use. Prior to use, bring these reagents
    to room temperature.

-   Vortex and briefly centrifuge the **Amplifier** and **Label Probe**
    to bring the contents to the bottom of the tube before use.

-   Calculations below for the **Wash Buffer** include a 20% overage.
    Calculations for the **Amplifier, Label Probe** **and Substrate
    Working Reagents** include a 10% overage. Determine the specific
    volume requirements for your system.

+-----+----------------------------------------------------------------+
| **  | **Action**                                                     |
| Ste |                                                                |
| p** |                                                                |
+-----+----------------------------------------------------------------+
| **  | Prepare the **Wash Buffer**:                                   |
| 1** |                                                                |
|     | a.  Combine the reagent volumes appropriate for the number of  |
|     |     plates used in the assay.                                  |
|     |                                                                |
|     | b.  Stir gently to mix.                                        |
|     |                                                                |
|     | c.  Keep mixture at room temperature.                          |
|     |                                                                |
|     | +---------------+-----------+-----------+-----------------+    |
|     | | **Capture**   | **10X     | **Di      | **Total**       |    |
|     | |               | bDNA**    | stilled** |                 |    |
|     | | **Plates**    |           |           | **Volume**      |    |
|     | |               | **Wash    | **H~2~0** |                 |    |
|     | |               | Buffer**  |           |                 |    |
|     | +---------------+-----------+-----------+-----------------+    |
|     | | 1             | 40 ml     | 360 ml    | 400 ml          |    |
|     | +---------------+-----------+-----------+-----------------+    |
|     | | 2             | 80 ml     | 720 ml    | 800 ml          |    |
|     | +---------------+-----------+-----------+-----------------+    |
+-----+----------------------------------------------------------------+
| **  | Prepare the **Amplifier Working Solution**:                    |
| 2** |                                                                |
|     | a.  Combine the reagent volumes appropriate for the number of  |
|     |     plates used in the assay.                                  |
|     |                                                                |
|     | b.  Stir gently to mix.                                        |
|     |                                                                |
|     | c.  Keep mixture at room temperature.                          |
|     |                                                                |
|     |   -                                                            |
|     | ------------------ ----------------------- ------------------- |
|     |   **Capture Plates**  **Amplifier/Diluent**   **Amplifier**    |
|     |                                                                |
|     |   1                   11.6 ml                 11.6 µl          |
|     |                                                                |
|     |   2                   22.0 ml                 22.0 µl          |
|     |   -                                                            |
|     | ------------------ ----------------------- ------------------- |
+-----+----------------------------------------------------------------+
| **  | Prepare the **Label Probe Working Reagent:**                   |
| 3** |                                                                |
|     | a.  Combine the reagent volumes appropriate for the number of  |
|     |     plates used in the assay.                                  |
|     |                                                                |
|     | b.  Stir gently to mix.                                        |
|     |                                                                |
|     | c.  Keep mixture at room temperature.                          |
|     |                                                                |
|     |   -                                                            |
|     | ------------------ ----------------------- ------------------- |
|     |   **Capture Plates**  **Amplifier/Diluent**   **Label Probe**  |
|     |                                                                |
|     |   1                   11.6 ml                 11.6 µl          |
|     |                                                                |
|     |   2                   22.0 ml                 22.0 µl          |
|     |   -                                                            |
|     | ------------------ ----------------------- ------------------- |
+-----+----------------------------------------------------------------+

D.  Prepare the Reagents

    Prepare the Reagents (cont.)

+-------+--------------------------------------------------------------+
| **S   | **Action**                                                   |
| tep** |                                                              |
+-------+--------------------------------------------------------------+
| **4** | Prepare the **Substrate Working Reagent**:                   |
|       |                                                              |
|       | a.  Combine the reagent volumes appropriate for the number   |
|       |     of plates used in the assay.                             |
|       |                                                              |
|       | b.  Stir gently to mix. It is normal for the **Substrate     |
|       |     Working Reagent** to become turbid after the addition of |
|       |     the **10% Lithium Lauryl Sulfate**.                      |
|       |                                                              |
|       | c.  Cover the solution with aluminum foil and keep at room   |
|       |     temperature.                                             |
|       |                                                              |
|       | +------------------+------------------+------------------+   |
|       | | **Capture        | **Substrate**    | **10% Lithium**  |   |
|       | | Plates**         |                  |                  |   |
|       | |                  |                  | **Lauryl         |   |
|       | |                  |                  | Sulfate**        |   |
|       | +------------------+------------------+------------------+   |
|       | | 1                | 11.6 ml          | 34.8 µl          |   |
|       | +------------------+------------------+------------------+   |
|       | | 2                | 22.0 ml          | 66.0 µl          |   |
|       | +------------------+------------------+------------------+   |
+-------+--------------------------------------------------------------+

> **E. Amplify and Detect the Target mRNA**

+------+---------------------------------------------------------------+
| **St | **Action**                                                    |
| ep** |                                                               |
+------+---------------------------------------------------------------+
|      |                                                               |
+------+---------------------------------------------------------------+
| *    | Wash the **Capture Plate:**                                   |
| *1** |                                                               |
|      | a.  Remove the plate from the incubator and remove the seal   |
|      |     from the plate.                                           |
|      |                                                               |
|      | b.  Add **250 µl** **Wash Buffer** to the wells then follow   |
|      |     one of the methods below.                                 |
+------+---------------------------------------------------------------+
| *    | Wash the **Capture Plate:**                                   |
| *1** |                                                               |
|      | c.  Remove the plate from the incubator and remove the seal   |
|      |     from the plate sealer.                                    |
|      |                                                               |
|      | d.  Add **250 µl** **Wash Buffer** to the wells then follow   |
|      |     one of the methods below.                                 |
+------+---------------------------------------------------------------+

> **Amplify and Detect the Target mRNA (cont.)**

+-------+--------------------------------------------------------------+
| **S   | **Action**                                                   |
| tep** |                                                              |
+-------+--------------------------------------------------------------+
| **2** | Immediately add 100 µl of **Amplifier Working Reagent** to   |
|       | each well.                                                   |
+-------+--------------------------------------------------------------+
| **3** | Reseal and incubate the plate at 46º to 53ºC for 60 minutes. |
+-------+--------------------------------------------------------------+
|       | a.                                                           |
|       | b.                                                           |
|       | c.                                                           |
+-------+--------------------------------------------------------------+
| **4** | Wash the **Capture Plate**:                                  |
|       |                                                              |
|       | d.  Remove the plate from the incubator and remove the seal. |
|       |                                                              |
|       | e.  Immediately aspirate or decant the **Amplifier Working   |
|       |     Reagent** from the wells and wash each well three times  |
|       |     with 350 µl of **Wash Buffer**.                          |
|       |                                                              |
|       | f.  Completely remove **Wash Buffer** from the wells. (Refer |
|       |     to Step 1.)                                              |
+-------+--------------------------------------------------------------+
| **4** | Wash the **Capture Plate**:                                  |
|       |                                                              |
|       | g.  Remove the plate from the incubator.                     |
|       |                                                              |
|       | h.  Immediately aspirate the **Amplifier Working Reagent**   |
|       |     from the wells and wash each well three times with 350   |
|       |     µl of **Wash Buffer**.                                   |
|       |                                                              |
|       | i.  Completely remove **Wash Buffer** from the wells. (Refer |
|       |     to Step 1.)                                              |
+-------+--------------------------------------------------------------+
| **5** | Immediately add 100 µl of **Label Probe Working Reagent** to |
|       | each well.                                                   |
+-------+--------------------------------------------------------------+
| **6** | Reseal and incubate the plate at 46º to 53ºC for 60 minutes. |
+-------+--------------------------------------------------------------+
|       | a.                                                           |
|       | b.                                                           |
|       | c.                                                           |
+-------+--------------------------------------------------------------+
| **7** | Wash the **Capture Plate**:                                  |
|       |                                                              |
|       | d.  Remove the plate from the incubator and remove the seal. |
|       |                                                              |
|       | e.  Immediately aspirate or decant the **Label Probe Working |
|       |     Reagent** from the wells and wash each well three times  |
|       |     with 350 µl of **Wash Buffer**.                          |
|       |                                                              |
|       | f.  Completely remove **Wash Buffer** from the wells. (Refer |
|       |     to Step 1.)                                              |
|       |                                                              |
|       | **Note: After hybridizing with Label Probe do not let the    |
|       | plate dry for more than 5 minutes to avoid partial           |
|       | inactivation of the AP activity.**                           |
+-------+--------------------------------------------------------------+
| **7** | Wash the **Capture Plate**:                                  |
|       |                                                              |
|       | g.  Remove the plate from the incubator.                     |
|       |                                                              |
|       | h.  Immediately aspirate the **Label Probe Working Reagent** |
|       |     from the wells and wash each well three times with 350   |
|       |     µl of **Wash Buffer**.                                   |
|       |                                                              |
|       | i.  Completely remove **Wash Buffer** from the wells. (Refer |
|       |     to Step 1.)                                              |
|       |                                                              |
|       | **Note: After hybridizing with Label Probe do not let the    |
|       | plate dry for more than 5 minutes to avoid partial           |
|       | inactivation of the AP activity.**                           |
+-------+--------------------------------------------------------------+
| **8** | Immediately add 100 µl of **Substrate Working Reagent** to   |
|       | each well.                                                   |
+-------+--------------------------------------------------------------+
| **9** | Reseal and incubate at 46º to 53ºC for 30 minutes.           |
+-------+--------------------------------------------------------------+
|       |                                                              |
+-------+--------------------------------------------------------------+
| *     | Remove the plate from the incubator and transfer the plate   |
| *10** | to a luminometer (without a plate heater) and allow the      |
|       | plate to cool for 5 minutes. **Alternatively**, for a        |
|       | luminometer with a plate heater, allow the plate to          |
|       | equilibrate in the tray for 5 minutes.                       |
+-------+--------------------------------------------------------------+
|       |                                                              |
+-------+--------------------------------------------------------------+
| *     | Measure the luminescence of each well.                       |
| *11** |                                                              |
+-------+--------------------------------------------------------------+