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.

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