Protocol for Reverse Transcription and Amino-allyl Coupling of RNA

The following is a slight modification* of a protocol developed by Joe DeRisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA). Original document can be obtained at www.microarrays.org.

A. RT Reaction

1. To anneal primer, mix 1-2 mg mRNA with 5 ug of anchored oligo-dT [(dT)₂₀-VN] (Operon, HPLC purified) in a total volume of 18 mL. One reaction for sample mRNA and one for reference mRNA.

oligo dT	5 mg of 2.5 mg/mL	2 mL
mRNA/water	1-2 mg	16 mL

2. Heat to 70C for 10 minutes. Cool on ice for 5 minutes.

3. Add 11.6 mL of nucleotide mix to each of Cy3 and Cy5 reactions.

Nucleotide Mix for one reaction

5X RT buffer 50X dNTP stock solution DTT Superscript II RT (Gibco) RNasin (Gibco, optional)

0.1M 200U/mL 40U/mL

6.0 mL 0.6 3.0 1.5 0.5

50X dNTP stock solution using a 4:1 ratio aminoallyl-dUTP to dTTP***:

10 mL each 100 mM dATP, dGTP, dCTP (Pharmacia)

8 mL 100 mM aminoallyl-dUTP** (Sigma, #A0410)

2 mL 100 mM dTTP

**Dissolve 10 mg aminoallyl-dUTP in 170 mL water. Add approx. 6.8 mL 1N NaOH. Final pH is roughly 7.0 using pH paper.

***Altering the ratio of aminoallyl-dUTP to dTTP will affect the incorporation of Cy dye.

1X dNTP final concentration during labeling

500 mM each dATP, dCTP, dGTP

400 mM aminoallyl-dUTP

100 mM dTTP

4. Incubate reaction for 1 hour at 42C. Add additional 1 mL reverse transcriptase and continue incubation at 42C for an additional 1 hour.

B. Hydrolysis

1. Degrade RNA by addition of 15 mL of 0.1 N NaOH. Incubate at 70C for 10 minutes

2. Neutralize by addition of 15 mL 0.1 N HCl.

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

3. Add 450 mL water to each reaction.

C. Cleanup

Add 500 mL neutralized, diluted reaction mix to a Microcon-30 filter (Amicon).

Spin at 12g for 7 minutes.

Discard flow through.

Repeat process two more times, refilling original filter with 450 mL water. Concentrate to 10 mL. Samples can be stored at -20C indefinitely.

D. Coupling

Add 0.5 mL 1M sodium bicarbonate, pH 9.0 to 50 mM final. Check 1M stock solution periodically for fluctuations in pH.

Monofunctional NHS-ester Cy3 (PA23001) and Cy5 dye (PA25001, Amersham) is supplied as a dry pellet. Each tube is sufficient to label 10 reactions under normal conditions. Dissolve dry pellet in 20 mL DMSO. Aliquot 2 mL into 10 single use tubes that are then dried in vacuo and store desiccated at 4C. NHS-ester conjugated Cy dye is rapidly hydrolyzed in water, therefore, do not store in DMSO or water. Decreasing the number of aliquots/dye tube may increase your signal.

If you have already made aliquots of dye, simply transfer your cDNA in bicarbonate buffer (10.5 mL) to the aliquot of dye. Alternatively, dissolve Cy dye in 10 ul DMSO and add 1 mL of dye to 10.5 mL of the cDNA reaction. 10% DMSO in the coupling reaction will not affect the chemical reaction. Aliquot unused dye and dry immediately.

Incubate 1 hour at RT in the dark. Mix every 15 minutes.

E. Quenching and Cleanup

Before combining Cy3 and Cy5 samples for hybridization, unreactive NHS-ester Cy dye must be quenched to prevent cross coupling.

Add 4.5 mL 4M hydroxylamine (Sigma).

Let reaction incubate 15 minutes in the dark.

To remove unincorporated/quenched Cy dyes, proceed with Qia-Quick PCR purification kit (QIAGEN). Method described below is as specified by manufacturer.

Combine Cy3 and Cy5 reactions.

Add 70 mL water.

Add 500 mL Buffer PB.

Apply to Qia-quick column and spin at 13K for 30-60 seconds. (optional: reapply flow-though for optimal binding).

Decant flow-through.

Add 750 mL Buffer PE and spin 30-60 seconds.

Decant flow-through.

Spin at high speed to dry column.

Transfer spin unit to fresh eppendorf tube.

Add 30 mL Buffer EB to center of filter and allow to sit 3 minutes at RT.

Spin at 13K rpm for 1 minute.

Repeat elution step again with another 30 mL of Buffer EB.

Pool eluates.

Add 20 mL (20 mg) Cot DNA (Gibco).

Add 420 TE and apply to fresh Microcon-30 filter.

Spin 12,000g to a volume of 29 mL or less.

For 38 mL array hybridization:

29 mL cDNA probe in TE

1 mL polyA (10 mg; Sigma P9403)

1 mL tRNA (10 mg; Gibco #15401-029)

7 mL 20X SSC

1.2 mL SDS 10%

Heat to 100C for 2 minutes. Let stand 15 minutes RT.

Apply 38 mL to 40K array.

* Slight modifications to original protocol by Mitch Garber and Anatoly Urisman.