Preparation of Epoxy Microarray Slides for Hybridization

1. Wash slides prior to hyb to remove unbound probe:
1. Etch the under side of the slide to indicate the edges of the printed samples (the side with the barcode contains the printed DNA)
2. Rinse 1 X 5’ with 0.1% Triton X-100 at rt
3. Rinse 2 X 2’ with 1mM HCL at rt
4. Rinse 1 X 10’ with 100mM KCL at rt

Each of these washes should be performed with 1 liter of solution and use the pyrex round glass dish with a stir bar on the bottom for constant agitation during the wash

2. Denature PCR probes
1. Boil with ddw 1 X 3’, use round pyrex dishes, the other ones will crack with heat
2. Cool in rt ddw 1 X 1’

3. Sodium borohydride treatment to decrease autofluorescence of the background
1. Make blocking solution and prewarm to 42^oC (use the square glass dish in a water bath, make sure sodium borohydride is dissolved, it takes about 5 minutes with stirring, this will evolve H₂ gas so do not use near flames, and when done, leave the solution in the hood for 24 hr before flushing down the drain with lots of water)
2. Place slides in block solution at 42^oC, soak for 20’ (400 mls, square glass dish)
3. Transfer to 1X SSC at rt to cool for 5’ (use ~400 mls in a square glass dish)
4. Wash 4X with 1X SSC for 5’ at rt (1 liter per wash with vigorous agitation, use round pyrex dish for these)
5. Wash 4X with 0.2 X SSC for 2 ; at rt (as above)
6. Wash 1 X with ddw for 2’ at rt
7. Place each slide in a 50 ml centrifuge tube and spin dry the slides at 200 X g for 5’ (place in the tube with the barcode up and retrieve using forceps)
8. store slides under argon or nitrogen gas for hours until ready for hyb.

4        Blocking, this step is important in preventing residual borohydride from quenching the fluorescent signals from the dyes used in hybridization.

a.       Incubate slides at 50^oC in 4% BSA +0.5% SDS (use a coplin jar for this to reduce the volume needed, in the hyb oven.  One Coplin jar will hold ~70 mls and 5 slides) for 1 X 15’

b.      Rinse in ddw 1 X 1’

c.       Dry slides in the centrifuge at 200 X g for 5’

d.      Slides now ready for hybridization

Microarray Hybridization

1.      Preparation of cDNA

a.       Mix the samples going into each hybridization

b.      Dry the sample down to 10 ml in the speedvac (do not dry completely)

c.       Add to each sample:  1M HEPES pH=7.8              1 µl

20 X SSC                           6 µl

Formamide                         20 µl

sterile ddw                          2 µl

20% SDS                           1µl

                                           40 µl

Make sure to add the SDS last and separately from other reagents.  Don’t mix excessively or you’ll get bubbles which are then trouble when you put the coverslip on.  Heat the probe to 95-100^oC for 2 min to denature, ice, spin down briefly to collect, now ready for hybridization.

For humidification of the chamber during hybridization:  make 6 mls 3 X SSC+50% formamide, saturate 2 kimwipes and place at the bottom of the slide chamber

2.      Carefully place the 40 µl of labeled cDNA on top of the array slide and lower the coverslip slowly to avoid bubbles.  I like to put one edge down and adjust its position relative to the etch mark and then slowly lower the other edge of the coverslip into place.  Place all slides in the microscope slide chamber and seal the box with gel sealing tape.

3.      Hybridize o/n at 50^oC submerged in a water bath.

4.      Washing the arrays

a.       1 X 10’ with 2 X SSC +0.2% SDS at rt

b.      1 X 10’ with 2 X SSC at rt

c.       1 X 10’ with 0.2 X SSC at rt

d.      spin dry at 200 X g, ready for scanning, store slides in the dark under argon or nitrogen if not ready to scan

To dislodge the coverslip, hold the array slide horizontally in the first buffer listed above and swish back and forth vigorously until the coverslip comes off, do not attempt to pry it off manually or you might damage the surface of the array.

For each of the washes use round glass pyrex dish with a stir bar on the bottom for vigorous mixing, 1 liter per wash.

Cover slides waiting to be scanned with foil to prevent exposure to light.

0.1% Triton X 100:

1ml of Triton X 100 +999 mls ddw

1mM HCL:

            83 µl of 12 N HCL in 1 liter of ddw

100mM KCL:

            100 mls of 1 M KCL diluted with 900 mls ddw

Blocking solution for sodium borohydride treatment:

                                    400 ml

20 X SSC        40 mls                          This solution must be made fresh!

ddw                 359 mls                        This solution will evolve H₂ gas

20% SDS         1 ml.

NaBH4 caplet  1 g ( 1 caplet)

This solution will be very foamy with gas evolution and SDS.  Leave in the hood for at least 24 h before discarding and flush down the drain with lots of water.

1X SSC:

                                                400 mls

20 X SSC                    20 mls

ddw                             380 mls

                                                1 liter

20 X SSC                    50 mls

ddw                             950 mls

0.2 X SSC:

                                                1 liter

20 X SSC                    10 mls

ddw                             990 mls

4% BSA + 0.5% SDS:

Add 4g BSA (fraction V) and 2.5 mls of 20% SDS to total volume of 100 mls with ddw warm to 50^oC

3 X SSC +50% formamide:

6mls

20 X SSC                    900 ml

formamide                    3 mls

ddw                             2.1 mls

Make up in a centrifuge tube and dip kimwipes in to saturate

Wash solutions:

                                    2 X SSC + 0.2% SDS                         1 liter

                                                20 X SSC                                100 mls

                                                20% SDS                                 10 mls

                                    0.2 X SSC                                           1 liter

                                                20 X SSC                                10 mls