SP, Jul 05
Reverse transcription is the reverse transcriptase-mediated synthesis of single-stranded DNA (complementary DNA or cDNA) using single-stranded RNA as template; the cDNA product and the template RNA have complementary sequence and can exist as RNA-DNA hybrid; the RNA, when in this hybrid state, is susceptible to degradation by RNAse H.
Any RNA - total RNA, mRNA, specific RNA, in vitro transcribed RNA - can be reverse transcribed as long as a single-stranded DNA primer is hybridized to the RNA; the reverse transcriptase enzyme will start synthesizing DNA from the 3' end of the primer with the RNA sequence acting as template. An oligo-dT primer (T18-24) will allow cDNA synthesis from almost all mRNA as it can bind to the poly-A tails found in most mRNA molecules. On the other hand, one can use a gene-specific primer to reverse transcribe specific mRNA.
Commonly used reverse transcriptases are viral products (e.g., the AMV reverse transcriptase of avian myeloblastosis virus). Engineered reverse transcriptases have higher processivity and lower error rates, and can function at higher temperatures that allows for better reverse transcription by resolving RNA folds and reduces non-specific reverse transcription as non-specific binding by the DNA primer is reduced.
Reverse transcription reaction product can be used as template for PCR, for generating cDNA library, etc. The single stranded cDNA can be converted to double stranded DNA using other polymerases, but this is not required for PCR. Note that reverse transcriptase enzyme left in the reaction can inhibit PCR. Also, PCR efficiency can be improved by treating the reaction with RNAse H beforehand (see above).
Follow protocols suggested by the manufacturer of the reverse transcriptase enzyme that you are using. Following is a typical method:
1. Use 0.01-2 poly-A+ RNA (mRNA) 1-5 ug total RNA per reaction of final 20 ul volume.
2. Add 0.5 ng (2 pmole) oligo-dT primer or random-hexamers (dN6), or 2-3 pmole gene-specific primer.
3. Add DEPC-treated or RNAse-free water of appropriate volume and heat at 70 °C for 10 min. Then chill immediately on ice. This step unfolds RNA secondary structures.
4. Add 0.1 M DTT for 10 mM final concentration.
5. Add dNTPs for final 0.5 mM concentration of each dNTP.
6. Add reaction buffer supplied by manufacturer.
7. Warm reaction to 42 °C, and after 2-3 min, add reverse transcriptase enzyme.
8. Incubate at 42 ° for 30-60 min for reverse transcription to proceed. For higher specificity and better resolution of RNA secondary structures, one can use up to 50 °C with Superscript II™ enzyme and up to 60 °C with the Superscript III™ enzyme. DMSO added to a final concentration of 2-7% can also help resolve RNA secondary structures.
9. Stop reaction and denature the enzyme by heating at 70 ° for 10-15 min.
10. Cool down the reaction and add 2 U RNAse H (usually from E. coli) and incubate at 37 °C for 20 min. You can freeze the reaction and do the RNAse H treatment later.
11. Freeze the reaction or use as template in PCR - use 5-10% of the reaction per PCR.