The usual precautions for avoiding degradation of RNA should be taken until the reverse transcription reaction is complete.
1. Make solution of 1 µg poly(A) RNA or 10 µg total RNA in 9 µl of 1X annealing buffer. This may require ethanol precipitating the RNA, washing with 70% EtOH, drying the pellet, and adding 9 µl 1X buffer. Or, if your starting solution of RNA in water is concentrated enough, you can simply mix the RNA, some 2X buffer, and water in the correct proportions.
2. Add 1 µl of the oligodeoxynucleotide primer:
100 pmol/µl for a specific 3' primer, 1000 pmol/µl for random hexamers
3. Heat 3 minutes at 65° and allow to cool to room temperature.
4. Make up some cDNA synthesis mix, and add 15 µl to each 10 µl annealed RNA sample.
5. Allow the reverse transcriptase reaction to go for 1 hour at 37° C.
When random hexamers are used, start the reaction at room temperature for 10 minutes.
6. Heat inactive for 10 minutes at 75°.
7. Use 2 µl of this cDNA mixture as a template for each 50 µl PCR amplification reaction, following standard procedures.
1X Annealing buffer:
250 mM KCl
10 mM Tris pH 8.3
1 mM EDTA
2X cDNA buffer:
48 mM Tris pH8.3
32 mM MgCl2
cDNA synthesis mix:
for each 15 µl reaction:
2X cDNA buffer 7.5 µl
100 mM DTT 1.25 µl
RNAse Block (Stratagene) 1 µl =40 U
or other RNAsin
1.25 mM dNTPs 5 µl
M-MuLV reverse transcriptase 0.25 µl = 6 U (Biolabs)
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