1.  **Purpose -** The purpose of this procedure is to describe the
    processes for preparing the RNA stocks for the MAQC Main Study.

2.  **References, abbreviations & definitions**

    1.  SUHRR: Stratagene Universal Human Reference RNA

    2.  HBRR: Ambion First Choice Human Brain Reference RNA

    3.  Nanodrop User's Manual

    4.  Agilent Bioanalyzer User's Manual

    5.  Agilent Bioanalyzer Reagent Kit Guides (RNA 6000 Nano)

    6.  MAQC: Microarray Quality Control project, representing a
        consortium of over 30 organizations.

3.  **Required equipment and reagents**

    1.  **Equipment**

+-----------------------------------------------------------------------+
| **ITEM**                                                              |
+-----------------------------------------------------------------------+
| # Heat Block or Water Bath set to 37°C                                |
+-----------------------------------------------------------------------+
| # Pre-labeled 0.5 ml                                                  |
| , screw cap, o-ring tubes (Axygen part #SCT-060-SS-L-C or equivalent) |
+-----------------------------------------------------------------------+
| # Refrigerated Microcentrifuge set to 4°C                             |
+-----------------------------------------------------------------------+
| # Agilent 2100 Bioanalyzer                                            |
+-----------------------------------------------------------------------+
| # NanoDrop spectrophotometer or equivalent                            |
+-----------------------------------------------------------------------+
| # Ice bucket trays                                                    |
+-----------------------------------------------------------------------+
| # Speed-vacuum                                                        |
+-----------------------------------------------------------------------+
| # Tube racks for 0.5ml screw-caps                                     |
+-----------------------------------------------------------------------+
| # Pre-labeled freezer storage boxes                                   |
+-----------------------------------------------------------------------+
| # -20°C Freezer (non-frost free)                                      |
+-----------------------------------------------------------------------+
| # Ambion, Inc. product boxes                                          |
+-----------------------------------------------------------------------+

2.  **Reagents**

+------------------------------------+---------------------------------+
| **ITEM**                           | **1^o^ Source**                 |
+------------------------------------+---------------------------------+
| # Crushed ice                      | laboratory                      |
+------------------------------------+---------------------------------+
| # RNA Storage Solution             | Ambion, Inc. Catalog \# 7000    |
+------------------------------------+---------------------------------+
| # RNAse-free water                 | Ambion, Inc. Catalog \# 9932    |
+------------------------------------+---------------------------------+
| # 100% Ethanol                     |                                 |
+------------------------------------+---------------------------------+
| # RNA 6000 Nano Assay kit          | Agilent \# 5065-4476            |
+------------------------------------+---------------------------------+
| # RNA 6000 Ladder                  | Ambion, Inc. Catalog #7152      |
+------------------------------------+---------------------------------+
| # SUHRR                            | Stratagene, lot #1130623        |
+------------------------------------+---------------------------------+
| # SUHRR RNAse-free water           | Stratagene, Catalog #740000-42  |
+------------------------------------+---------------------------------+
| # HBRR                             | Ambion, lot# 055201             |
+------------------------------------+---------------------------------+

4.  **Stock solutions to be made**

    1.  70% Ethanol

        1.  Add 28 ml 100% Ethanol and 12 ml RNAse-free water into a
            50-ml conical screw-cap tube.

        2.  Mix thoroughly.

        3.  Store at -20°C

5.  **RNA Preparation**

    1.  SUHRR needs to be precipitated, washed and dissolved in
        RNase-free water provided with RNA.

        1.  Centrifuge all SUHRR tubes in the microfuge at 4°C for 15-30
            minutes at ≥12000Xg.

        2.  Carefully remove ethanol from precipitated pellet with
            RNase-free technique and RNase-free tips.

        3.  Add 1 ml cold 70% ethanol to each tube.

        4.  Mix thoroughly by vortexing.

        5.  Centrifuge all SUHRR tubes in the microfuge at 4°C for 15
            minutes at ≥12000Xg.

        6.  Carefully remove ethanol from precipitated pellet with
            RNase-free technique and tips.

        7.  Dry remaining ethanol at room temperature open to air for 30
            minutes.

        8.  Dissolve each of the pellets in 190μl RNAse-free water
            provided with RNA. Mix thoroughly by vortexing each tube for
            a minimum of 30 seconds. .

        9.  Allow at least 30 minutes for the RNA to thoroughly
            dissolve.

        10. Take Nanodrop reading of one tube. The concentration should
            be 1 ug/ul +/- 10% (0.9-1.1 ug/ul). If the concentration is
            too low, place the tubes at 37°C for 10 minutes, vortex
            thoroughly, and test the concentration of another sample.

        11. Mix all dissolved SUHRR samples by combining them in a
            RNase-free, sterile 50ml conical tube with screw cap.

    2.  Thaw the stock of HBRR.

        1.  Place the sample in a 37°C hot plate.

        2.  Manually mix the sample throughout the thawing time to avoid
            the formation of "warm spots" at the interface with the
            tube.

        3.  When ice is thawed completely, place the tube on the ice
            bath.

        4.  Place tube on the ice for 15-30 minutes. After 5 minutes,
            vortex for at least 30 seconds to ensure homogenous mixing
            of the RNA.

6.  **Procedure**

    1.  Ensure that all manipulations are performed with meticulous
        RNAse-free pipetting and handling techniques and RNA is stored
        on ice throughout the procedure. Only adequately trained and
        qualified personnel will handle the RNA.

    2.  Assess RNA concentrations for each stock SUHRR and HBRR using
        the Nanodrop.

        1.  Blank the samples with RNA Storage buffer.

        2.  Take at least 3 independent readings.

    3.  Calculate the dilution needed to bring the concentration of the
        more concentrated RNA stock to within 5% of other RNA stock.

    4.  Add the volume of RNA storage buffer calculated in step 6.3 to
        the more concentrated RNA sample.

    5.  Repeat steps 6.2-4, if necessary until both RNA stock
        concentrations are within 5% of each other.

    6.  Record the following from the Nanodrop for each RNA stock:

        1.  OD 260

        2.  260/280

        3.  260/230

        4.  Trace of the wavelength scan

    7.  Create mixtures of the HBRR and SUHRR stock RNAs according to
        the table:

  ------------------------------------------ -------------- -------------
  Sample name                                SUHRR volume   HBRR volume
                                             (ml)           (ml)

  Mix C MAQC (25% HBRR / 75% SUHRR)          **TBD**        TBD

  Mix D MAQC (75% HBRR / 25% SUHRR)          TBD            TBD
  ------------------------------------------ -------------- -------------

1.  Mix the RNA samples thoroughly; store on ice.

2.  Verify the expected final volume of each mixture. **\_\_\_\_\_\_\_\_
    volume**

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8.  Distribute the RNA into 60 labeled tubes.

    1.  Set up the appropriate number of tubes in racks on ice

    2.  Separate the tubes for each of the four sample types to be
        dispensed separately.

    3.  Dispense 50μl of the appropriate RNA into each of the labeled
        tubes

    4.  Cap tubes securely.

    5.  Place all tubes in the appropriate freezer box

9.  Quality Control assessment of the RNA after packaging

    1.  Submit one set of 4 tubes to Ambion for the following testing:

        1.  Overnight stability testing

        2.  Nuclease testing

    2.  RNA quality assessment on Agilent Bioanalyzer. Record 28S/18S
        ratio and RIN

10. Package sets of four RNA samples in an Ambion product box for
    shipping

    1.  Open freezer boxes of tubes are placed in dry ice for the
        transfer

    2.  Packaged sets are stored in the specified -80°C freezer

> Freezer\_\_\_\_\_\_\_\_\_
>
> Rack\_\_\_\_\_\_\_\_\_\_\_
>
> Slot\_\_\_\_\_\_\_\_\_\_\_\_

7.  Quality Control specifications

    1.  28S/18S ribosomal ratio greater than or equal to 1.0

    2.  Overnight stability: no more than 20% deterioration after
        overnight storage at 37°C

    3.  Nuclease testing.