Lance D. Miller

NIH-NCI-DCS-MSO

(adapted from J. DeRisi;

Pat Brown Lab, Stanford)

Probe Preparation/Hybridization Using PolyA RNA

reagents

PolyA RNA, 1-2 µg

Oligo dT_12-18, 0.5 µg/µl; GIBCO/BRL (or custom made, cartridge-purified 20mers at 2.0 mg/ml)

SuperScript II RT, 200 U/µl; GIBCO/BRL

5X 1^st Strand Buffer; GIBCO/BRL

0.1M DTT; GIBCO/BRL

50X dNTPs, (25 mM each of dA, dG, dC and 10 mM of dT); PHARMACIA

Cy3-dUTP; Amersham

Cy5-dUTP; Amersham

TE, (10 mM Tris pH 8.0 and 1 mM EDTA)

Microcon 30 spin columns; Amicon

human COT-1 DNA, concentrated to 10 µg/µl; Boehringer Mannheim

polyA, concentrated to 8 µg/µl; Pharmacia

20X SSC; GIBCO/BRL

10% SDS

sterile dH₂O

A. Reverse Transcription Setup

1. In a sterile microfuge tube, combine 3 µl Oligo dT (6 µg), sterile dH₂O, and 1-5 µg polyA RNA for a total volume of 15.5 µl.
2. Incubate @ 70^oC for 10 minutes then chill on ice.
3. Make cocktail as follows:

reagent	Per sample		X 5 samples
5X buffer		6 µl		30 µl
50X dNTPs		0.5 µl		2.5 µl
0.1M DTT		3 µl		15 µl

note: always make enough for 1 extra sample to compensate for pipetting error.

4. Add 9.5 µl aliquots of cocktail to each sample.
5. Add 3 µl of the appropriate Cy -dUTP and 2 µl SuperScript II RT to each sample.
6. Incubate @ 42^oC for 30 minutes, add 2 µl more SSII RT, and incubate another 30 minutes.

B. Probe Cleanup

7. Add 500 µl TE to each probe sample and transfer to Microcon 30 concentrators.
   note: prior to use, spin 500 µl TE through each Microcon column to remove glycerol from membrane.
   (At this time, turn on dri-bath (tempblock) set @ 100^oC.)
8. Spin samples @ 14000 xg (13000 rpm on eppendorf 5415C) for 8 minutes, check volume. Continue spinning as necessary until volume is reduced to 10-30 µl.
   note: minimize contact between pipette tip and Microcon membrane as membrane is fragile.
9. On one of the columns, combine the appropriate Cy3 and Cy5 probes (10-30 µl each), and add 450 µl TE. Reduce volume as before to 10-30 µl. (At this step, magenta-colored probe indicates good incorporation of both fluors.)
10. Wash the probe a final time (to remove unincorporated fluor) by adding 450 µl TE and reduce volume to 7 µl.
11. Invert column into a fresh microfuge tube (provided with kit) and spin 1 minute at maximum speed to recover probe. (If your volume is < 9 µl, bring volume up to 9 µl with TE.)
12. For a final volume of 15 µl, add 2 µl (20 µg) human COT-1 DNA, 1 µl (8-10 µg) polyA, 2.6 µl 20X SSC ( or 3.5X final), and 0.45 µl 10% SDS ( or 0.3% final).
    OPTIONAL: Before adding SDS, put through 0.2 µm spin filter to remove any trace particulates.
13. Heat for 1 minute at 100^oC.
14. Spin 10 minutes at 14000 xg to pellet any particulate matter and to facilitate cooling.
15. Let probe cool at room temperature for at least 15 minutes.

C. Hyb Setup

16. Add total volume of probe (~15 µl) to the center of a processed array and quickly (but carefully!) add cover slip (22mm x 22mm) taking care to prevent bubble accumulation beneath slip.
    Note: use forceps to lower cover slip and gently maneuver to spread probe evenly over array.
17. Place slide in hyb chamber, add 10 µl 3X SSC to far end of slide (to maintain humidity), firmly attach margin clamps, and incubate overnight (10-16 hours) @ 65^oC.

D. Hyb Washes

18. Carefully remove margin clamps of hyb chamber to prevent water from seeping in and contaminating the array.
19. Remove slide from chamber, place in slide rack, and submerge in staining dish filled with 2X SSC and 0.1% SDS. Plunge gently until cover slip falls away from the slide. (Tilt slide at an angle as you plunge to ensure cover slip falls away from array without scratching it.)
20. Wash for 1 minute in 1X SSC, plunging gently.
21. Wash for 1 minute in 0.2X SSC, plunging gently.
22. Wash for ~10 seconds in 0.05X SSC, plunging gently.
23. Quickly spin dry (in slide rack) at 550 rpm (Beckman GS-6KR) for 3-4 minutes.
24. Scan as soon as possible (scan Cy5 (red) channel first since it is prone to fading).

WASHES:	2XSSC+0.1%SDS	1XSSC	0.2XSSC	0.05XSSC
dH20:	179 ml	190 ml	198 ml	200 ml
20XSSC:	20 ml	10 ml	2 ml	0.5 ml
20%SDS:	1 ml	-	-	-