+-----------------------------+-------------------+-------------------+
| Recommended Supplies for    |                   |                   |
| Microarray Labeling 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 (RNase H)    | Invitrogen        | 18021-071         |
+-----------------------------+-------------------+-------------------+
| Formamide,                  | Any reputable     | We recommend      |
|                             | chemical          | buying small      |
| molecular biology grade,    | manufacturer      | aliquots, ≤ 100ml |
|                             |                   | bottles           |
| (deionized if available)    |                   |                   |
+-----------------------------+-------------------+-------------------+
| Coverslips for 48-pin       | Erie Scientific   | 25X60I-2-4789     |
| print:                      |                   |                   |
|                             | Erie Scientific   | 25X60I-M-5439     |
| Lifterslips™ (25 X 60 mm)   |                   |                   |
|                             |                   |                   |
| or mSeries™ (25 X 60 mm)    |                   |                   |
+-----------------------------+-------------------+-------------------+
| Staining Dish/rack (10      | Fisher            | 08-812            |
| slide)                      |                   |                   |
+-----------------------------+-------------------+-------------------+
| 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.,    | AHC               |
| >                           | Inc.              |                   |
| > Single Hyb Chamber        |                   | 2551              |
| >                           | Corning           |                   |
| > Single Hyb Chamber        |                   | AHCXD             |
|                             | Telechem Int.,    |                   |
| Deeper hyb chamber to       | Inc.              |                   |
| 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   | Invitrogen        | 15561-020         |
| BSA                         |                   |                   |
+-----------------------------+-------------------+-------------------+
| Centrifuge with microplate  |                   |                   |
| carrier assembly            |                   |                   |
+-----------------------------+-------------------+-------------------+

**[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.  For each dye dilute total RNA to between 5.0 -- 20.0 µg in 16.0µl of
    DEPC water (0.3- 1.25 µg/µl). 

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

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

3.  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.

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5.  Add 2 µl of 400 U/µl SuperScript™ III RT and incubate at 48°C for
    2hrs . (Final volume is 30µl)

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

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

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

9.  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 200 μl of Binding buffer ***PB*** to each RT reaction and mix
    well.

> [Note]{.underline}: recommended maximum is 2 RT reactions per column.

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

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2.  Spin for 1 min at full speed.

3.  Discard flow-through.

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

5.  Spin for 1 min at full speed.

6.  Discard flow-through.

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

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

9.  Add 10μl of 1:10 Elution buffer ***EB*** directly to the membrane.
    (dilute elution buffer 1:10 with Molecular Biology Grade water).

10. Incubate for 1 min. at room temperature.

11. Spin for 1 min at full speed.

12. Add another 10μl of diluted ***EB*** buffer to the membrane.

13. Incubate for 1 min. at room temperature.

14. Spin for 1 min at full speed.

15. Dry down in SpeedVac for \~15 min at medium temp.  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.)

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

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

3.  Mix thoroughly by gently pipetting up and down.

4.  Incubate minimum for 30 minutes 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 labled cDNA reaction,
    mix well.

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1.  Add 200μl of Binding buffer ***PB*** to each reaction and mix well.

2.  Apply each reaction to a separate spin column.

3.  Incubate for 1 min. at room temperature.

4.  Spin for 1 min at full speed.

5.  Discard flow-through.

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

8.  Spin for 1 min at full speed.

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8.  Discard flow-through.

9.  Repeat wash step.

10. Discard flow-through.

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

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

13. Add 10μl of 1:10 diluted Elution buffer ***EB*** directly to the
    membrane

14. Incubate for 1 min. at room temperature.

15. Spin for 1 min at full speed.

16.  Add another 10μl of 1:10 diluted Elution buffer ***EB*** directly
    to the membrane.

17. Incubate for 1 min. at room temperature.

18. Spin for 1 min at full speed.

19. Can read OD with the 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 80 μl of pre-hybridization buffer under a Lifterslip to the
    array and incubate for 42° C for at least 30 mins and up to 1 hour. 

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1.  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.

2.  Allow slide to air dry completely prior to use or spin dry.

>  

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

1.  Combine Cy3 and Cy5 labeled targets together (\~20μl recovered for
    each).

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1.  Denature target at 100^o^C for 1 minute, then snap cool on ice.
    (Final volume should be about 40µl)

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

3.  Add 20μl of water to wells in hybridization chamber to maintain
    humidity.

4.  Add 40μl of 2X F-hyb buffer to samples

5.  Load 80μl sample onto microarray under a Lifterslip.

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

 

**[Wash:]{.underline}  **

•          2x SSC, 0.1% SDS for 2 minutes, with occasional plunging

•          1x SSC, for 2 minutes, occasional plunging

•          0.2x SSC, for 2 minutes, occasional plunging

•          Spin 3 minutes / 650 rpm to dry

**  **

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