Amino-allyl Reverse Transcription

Resuspend the contents of one pack of the monofunctional NHS-ester Cy3 or Cy5 dye (mono-functional Cy3 or Cy5 reactive pack, Amersham catalog PA 23001 and PA 25001) in 72 ul DMSO. Aliquot 16 x 4.5 ul and either use immediately or dry the aliquots in a speed vac. Store aliquots at 4 C in a dessicator. Use one aliquot of the dyes for each RT labelling reaction and microarray hybridization.

I. RT Reaction

Oligo dT/Random Prime RNA:

	Concentration	uL
Oligo dT:pdN6	5ug/uL	1
Total Vol. of RNA		14.5

Incubate RNA (1-4 microgram poly A+ RNA) and oligo dT at 70 C for 10 min.
Chill on ice 10 min.

1x dNTPS: 500uM each dATP, dCTP, dGTP, 200uM aadUTP, 300uM dTTP

50x recipe: FOR 2:3 10uL each 100mM dATP,dGTP,dCTP, 4uL 100mM aa-dUTP*, 6uL 100 mMdTTP

* 5-(3-Aminoallyl)-2'-deoxyuridine 5'-triphosphate sodium salt, Sigma-Aldrich Catalog A-0410

cDNA synthesis

	Concentration	uL	10.5
5x buffer		6	63
50x aa dUTP/dNTPs		0.6	6.3
DTT	0.1M	3	31.5
SuperScript II		1.9	19.95
Water		3	31.5
		14.5	14.5
42 degrees for 2 hours			Aliquots

II. Hydrolysis

Add: 10ul 1N NaOH

10ul .5M EDTA

Incubate: 15 min. at 65° C.

Neutralize: 25ul 1M Tris pH 7.4 or 1M HEPES pH 7.5

III. Cleanup
To continue with the amino-allyl dye coupling procedure all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cye dyes from coupling to free amine groups in solution.

Fill one Microcon 30 concentrator with 450 ul water.

Add neutralized reaction.

Spin at 12K for 8 minutes.

Dump flo-thru.

Repeat process 2X, refilling original filter.

Elute.

Dry eluate in speed vac.

IV. Coupling

Resuspend cDNA pellet in 9ul 0.1M NaBicarbonate Buffer pH 9.0. Add this to the dried aliquot of Cy3 or Cy5 dye. Mix dye and cDNA.

Let incubate 1 hour at RT in dark.

V. Quenching and Cleanup
Before combining Cy3 and Cy5 samples for hybridizations, the reactions must be quenched to prevent cross-coupling.

Add 4.5ul 4M hydroxylamine.

Let reaction incubate 15 min. at RT in dark.

To remove unicorporated/quenched cye dyes proceed with Qia-quick PCR purification kit.

Combine Cy3 and Cy5 reactions.

Add 70ul water.
Add 500ul Buffer PB.

Apply to Qia-quick column and spin at 13,000 rpm in for 30-60 sec.

Aspirate off flo-thru.

Add 750ul Buffer PE and spin 30-60 sec.

Aspirate off flo-thru and repeat.
Aspirate flo-thru and spin for 1 min. at high speed to dry column.

Transfer to fresh epp. tube

Add 30ul Buffer EB to center of filter and let sit 1 min. at RT.

Spin at 13,000 rpm for 1 min.

Repeat elution step again.

VI. Hyb. Prep.

Dry down Qia-quick eluate in speed vac.

Bring volume to 18 ul with water and HEPES pH 7.5 so that the final concentration (after SSC and other additions) is 25 mM.

Add: 3.6ul 20X SSC

1.8ul polyA(10mg/ml) and/or other competitor nucleic acid

Optional: filter in Millipore 0.45micron spin column.
Add: 0.54ul 10% SDS.

Incubate reaction at 100° C for 2 min.

Apply to prepared microarray.