####### [Target Preparation/Hybridization Using Total RNA]{.underline}

***I. [cDNA Generation:]{.underline}***

Prepare separate cDNA labeling reaction for each fluorescent dye you
wish to use.

A "master mix" (step 4) can be made, and the reaction increased up to 5X
if needed.

1.  Make dilution of 5.0 -- 20.0 µg of total RNA in 16.0µl of DEPC
    water.

2.  Add 2.0µl of 2.5μg/µl anchored oligo d(T)~20~ primer.

3.  Incubate at 70°C for 5 minutes. Cool on ice for at least 1 min.

4.  Combine the following components for each sample in a sterile,
    RNase/Dnase-free microcentrifuge tube:

    a.  6.0µl of 5X First-Strand buffer

    b.  1.5µl of 0.1 M DTT

    c.  1.5µl of 10mM dNTP mix

    d.  1.0µl of RNaseOUT™ (40 U/µl )

5.  Add the mixture to the annealed primer and RNA.

6.  Add 2µl of 400 U/µl SuperScript™ III RT and incubate at 48°C for
    2hrs.

7.  Incubate at 70°C for 5 minutes to stop reaction.

8.  Cool down by spinning in a microcentrifuge at maximum speed for 1
    minute.

9.  Add 2μl of 2 U/μl RNase H and incubate at 37°C for 20 min.

10. Add 0.5 μl of 0.5M, pH 8.0 EDTA, mix well and proceed with
    purification.

# *II. [cDNA purification:]{.underline}* (QIAGEN MINElute purification kit)

1.  Add 100 μl of Binding buffer ***PB*** to RT reactions and mix well.

> [Note]{.underline}: Can add a maximum of 2 reactions per column.

2.  Apply to separate spin columns. Incubate for 1 minute.

3.  Spin for 1 min at full speed.

4.  Discard flow-through.

5.  Add 500 μl of Wash buffer ***PE*** per reaction (Be sure that
    ethanol was added to ***PE*** buffer).

6.  Spin for 1 min at full speed.

7.  Discard flow-through.

8.  Repeat wash step.

9.  Discard flow-through.

10. Spin for 1 min at full speed to eliminate the possibility of
    carrying over Wash buffer.

11. Place columns in a fresh 1.5ml microcentrifuge tubes.

12. Add 10 μl of Elution buffer directly to the membrane. (20 μl of
    Elution buffer if 2 rxns were put on the column).

13. Incubate for 1 min.

14. Spin for 1 min at full speed.

15. Apply flow-through back on membrane.

16. Incubate for 1 min.

17. Spin for 1 min at full speed.

18. Discard columns, spec on Nanodrop to determine cDNA concentration.

19. Dry down in SpeedVac for 15 min at medium temp. DO NOT OVERDRY!

*II. [Alternative procedure -- cDNA precipitation]{.underline}:*

1.  Add 3µl of 3M sodium acetate, pH 4.5.

2.  Add 1µl of 20mg/ml glycogen.

3.  Add 100µl of ice-cold 95% EtOH.

4.  Incubate at --20°C for at least 30 minutes. The reaction can be
    stored at this point for several days or up to 2 weeks.

5.  Spin the reaction at 13-14,000Xg for 20 minutes at 4°C. Carefully
    decant supernatant.

6.  Wash with 0.5 ml ice cold 70% EtOH and spin at 13-14,000Xg for 15
    minutes at 4°C. Carefully decant supernatant and let to air dry. A
    vacuum dryer can be used but DO NOT OVERDRY!

***III. [NHS-ester containing dyes coupling reaction:]{.underline}***

1.  Resuspend cDNA pellet in 5µl of 2x coupling buffer. (If pellet was
    over dried gently heat at 37° C for 15 minutes to aid in the
    resuspension process.)

2.  The first time a tube of dye is used, resuspend in 45µl DMSO. Use
    DMSO provided with the kit.

3.  Add 5µl of the resuspended monofunctional reactive dye to cDNA.

4.  Mix thoroughly by gently pipetting up and down.

5.  Incubate for 30 minutes up to 1 hour at room temp in the dark,
    flicking the tubes occasionally.

***IV. [Dye-Coupled cDNA Purification:]{.underline}*** *(using QIAGEN
MINElute purification kit)*

1.  Add 10μl of 3M Sodium Acetate, pH 5.2 to each RT reaction, mix well.

2.  Add 100 μl of Binding buffer ***PB*** to RT reactions and mix well.

3.  Apply each RT reaction to separate spin columns.

4.  Incubate for 1 min.

5.  Spin for 1 min at full speed.

6.  Discard flow-through.

7.  Add 500 μl of Wash buffer ***PE*** per reaction (Be sure that
    ethanol was added to ***PE*** buffer).

8.  Spin for 1 min at full speed.

9.  Discard flow-through.

10. Repeat wash step.

11. Discard flow-through.

12. Spin for 1 min at full speed to eliminate the possibility of
    carrying over Wash buffer.

13. Place columns in a fresh 1.5ml microcentrifuge tubes.

14. Add 10 μl of Elution buffer directly to the membrane. (20 μl of
    Elution buffer for 48-pin print).

15. Incubate for 1 min.

16. Spin for 1 min at full speed.

17. Apply flow-through back on membrane.

18. Incubate for 1 min.

19. Spin for 1 min at full speed.

20. Can spec using Nanodrop to determine labeling efficiency and cDNA
    concentrations.

######### V. [Pre-hybridization:]{.underline} (should start approximately 2 hours before setting up hybridization)

Pre-hybridization buffer = 5X SSC, 0.1% SDS and 1% BSA. (Can make 10%
BSA stock and filter before use or purchase pre-filtered BSA; store
pre-hyb buffer at -20° C and thaw only once, warm to 42° C prior to
use.)

1.  Apply 40 μl of pre-hybridization buffer to the array and incubate
    for 42° C for at least 30 mins and up to 1 hour. (If a 48-pin print,
    then apply 80 μl of pre-hyb buffer to the array.)

2.  Wash off the pre-hybridization solution by rapidly plunging the
    slide in distilled water for 2 mins, then transfer slide to 100%
    isopropanol for 2 mins.

3.  Allow slide to air dry completely prior to use. (Can spin dry if in
    a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying
    before setting up hybridization.

## 

***VI. [Setting up hybridization:]{.underline}***

1.  Combine Cy3 and Cy5 labeled targets together (\~9 μl recovered for
    each).

2.  Add 1µl COT-1 DNA (8-10 µg/µl) and 1µl poly A (8-10 µg/µl).

3.  Denature target at 100^o^C for 1 minute, then snap cool on ice.
    (Final volume should be about 20µl.)

4.  Make fresh 2X Formamide hybridization buffer (50% formamide, 10x
    SSC, 0.2% SDS) and warm to 42^o^C just before adding to samples.

5.  Add 20μl of 3X SSC to wells in hyb chamber to maintain humidity.

6.  Add 20μl of 2X F-hyb buffer to samples. (Add 40 μl of 2X F-hyb
    buffer to samples for 48-pin print.)

7.  Load 40μl sample onto microarray. (Load 80 μl onto 48-pin print.)

8.  Incubate overnight (12-16 hours) at 42° C in water bath or
    hybridization oven.

Wash:

+-----------------------------------+----------------------------------+
| a\. **[cDNA                       | b[. **Oligo                      |
| slides:]{.underline}**            | slides:**]{.underline}           |
|                                   |                                  |
| -   2x SSC, 0.1% SDS for 2        | -   2x SSC, 0.1% SDS for 2       |
|     minutes, with occasional      |     minutes, with occasional     |
|                                   |                                  |
| > plunging                        | > plunging                       |
|                                   |                                  |
| -   1x SSC, for 2 minutes,        | -   1x SSC, for 2 minutes,       |
|     occasional plunging           |     occasional plunging          |
|                                   |                                  |
| -   0.2x SSC, for 2 minutes,      | -   0.2x SSC, for 2 minutes,     |
|     occasional plunging           |     occasional plunging          |
|                                   |                                  |
| -   0.05X SSC, for 1 minute       | -   [NOTE:]{.underline} The last |
|                                   |     wash of 0.05X SSC is omitted |
| -   Spin 3 minutes / 650 rpm to   |                                  |
|     dry                           | -   Spin 3 minutes / 650 rpm to  |
|                                   |     dry                          |
| ##                                |                                  |
+-----------------------------------+----------------------------------+

> **WASHES:** [2XSSC+0.1%SDS]{.underline} [1XSSC]{.underline}
> [0.2XSSC]{.underline} [0.05XSSC]{.underline}
>
> [dH~2~0:]{.underline} 179 ml 190 ml 198 ml 200 ml
>
> [20XSSC:]{.underline} 20 ml 10 ml 2 ml 0.5 ml
>
> [20%SDS:]{.underline} 1 ml - - -
>
> Rev 0305

## 

+-------------------------------+---------------------+----------------+
| Recommended Supplies for      |                     |                |
| Microarray Probe and          |                     |                |
| Hybridization                 |                     |                |
|                               |                     |                |
| (*March 2005)*                |                     |                |
+-------------------------------+---------------------+----------------+
| **SUPPLIES**                  | **SUPPLIER**        | **Catalog      |
|                               |                     | No.**          |
+-------------------------------+---------------------+----------------+
|                               |                     |                |
+-------------------------------+---------------------+----------------+
| SuperScript™ Indirect cDNA    | Invitrogen          | L1014-02       |
| Labeling Kit                  |                     |                |
+-------------------------------+---------------------+----------------+
| Dyes:                         | Pharmacia           | PA23001        |
|                               |                     |                |
| Cy3 monofunctional reactive   | Pharmacia           | PA25001        |
| dye                           |                     |                |
|                               |                     |                |
| Cy5 monofunctional reactive   |                     |                |
| dye                           |                     |                |
+-------------------------------+---------------------+----------------+
| Ribonuclease H                | Invitrogen          | 18021-071      |
+-------------------------------+---------------------+----------------+
| Poly (dA) 40-60               | Pharmacia           | 27-7988-01     |
+-------------------------------+---------------------+----------------+
| COT-1 DNA:                    | Invitrogen          | 15279011       |
|                               |                     |                |
| Human COT-1 DNA (for human    | Invitrogen          | 18440016       |
| arrays)                       |                     |                |
|                               |                     |                |
| Mouse COT-1 DNA (for mouse    |                     |                |
| arrays)                       |                     |                |
+-------------------------------+---------------------+----------------+
| Coverslips:                   | Erie Scientific     | 25X40I-2-4772  |
|                               |                     |                |
| 32-pin print: Lifterslips™    | Erie Scientific     | 25X40I-M-5227  |
| (25 X 40 mm)                  |                     |                |
|                               | Erie Scientific     | 25X60I-2-4789  |
| mSeries™ (25 X 40 mm)         |                     |                |
|                               | Erie Scientific     | 25X60I-M-5439  |
| 48-pin print: Lifterslips™    |                     |                |
| (25 X 60 mm)                  |                     |                |
|                               |                     |                |
| mSeries™ (25 X 60 mm)         |                     |                |
+-------------------------------+---------------------+----------------+
| Staining Dish/rack (10 slide) | Fisher              | 08-812         |
+-------------------------------+---------------------+----------------+
| Slide Box (100 slide)         | Thomas Scientific   | 6708-G28       |
+-------------------------------+---------------------+----------------+
| Slide Box (25 slide)          | Thomas Scientific   | 6708-G08       |
+-------------------------------+---------------------+----------------+
| Hybridization chambers:       | Genomic Solutions   | JHYB200004     |
|                               |                     |                |
| > Dual Hyb Chamber            | Telechem Int., Inc. | AHC            |
| >                             |                     |                |
| > Single Hyb Chamber          | Corning             | 2551           |
| >                             |                     |                |
| > Single Hyb Chamber          | Telechem Int., Inc. | AHCXD          |
|                               |                     |                |
| Deeper hyb chamber to         |                     |                |
| accommodate thicker mSeries™  |                     |                |
| cover slip:                   |                     |                |
|                               |                     |                |
| > Single Hyb Chamber          |                     |                |
+-------------------------------+---------------------+----------------+
| Hyb Oven                      | Fisher Scientific   | 13-247-10      |
+-------------------------------+---------------------+----------------+
| Forceps                       | Fisher Scientific   | 10-295         |
+-------------------------------+---------------------+----------------+
| Mini-Elute PCR Purification   | Qiagen              | 28004          |
| Kit                           |                     |                |
+-------------------------------+---------------------+----------------+
| High Quality Pre-filtered BSA | Invitrogen          | 15561-020      |
+-------------------------------+---------------------+----------------+
| Centrifuge with microplate    |                     |                |
| carrier assembly              |                     |                |
+-------------------------------+---------------------+----------------+