Strategic Planning and basic protocol: Currently everyonein the Szostak lab uses essentially the same protocol and products forreverse transcription. There are several other options, but they have notbeen explored by me or others in the group, so they are not discussed here.We use the GibcoBRL Superscript II Rnase H- Reverse Transcriptase, CatalogNo. 18064-014. The enzyme comes in 200U/ul and is supplied with a reactionbuffer ("5x First Strand Buffer") and 0.1M DTT. We follow the directionsas indicated in the product sheet. The only other reagent that we needto supply ourelves are the dNTP lab stocks. People sometimes add Rnasinat the suggested concentration.
Quantitative RT: Label the RT primer by kinasing with32P-gamma-ATP. Use a mixture of this hot primer and a known amount of coldprimer, say 2uM. Do the RT reaction exactly as indicated in the productsheet. After the reaction, take a sample, degrade all the RNA by bringingsample to 100mM NaOH, and heat at 90°C for 10 minutes. Then neutralize,add urea loading buffer, and run on a sequencing gel. The gel should beof a percentage that allows the RT primer to form a sharp band, but alsoallows the full-length product of the reaction enter the gel and be resolvedfrom other large RNAs. After running the gel, quantify the relative amountof radioactivity in the primer band and the product band. From this youwill know the concentration of RT product. If you know how much RNA youstarted with, you can determine the RT efficiency.
Pitfalls and troubleshooting: The RT reaction is delicate,and so it is important to prove to yourself that the reaction has indeedworked. The best way to do this is by doing a quantitative RT reaction(described above). A less quantitative way is to do PCR after the RT reaction,and try to use these results to quantify the amount of cDNA. For this lattermethod to be trusted, it is very important to do a control RT reactionwitout RT enzyme. This controls for the presence of DNA contamination,which can be a major problem in selections or any other source of RNA thatwas made by transcription.
One potential source of failed RT reactions is that the RT reactioncontains only 3mM Mg++. Thus, small amounts of EDTA (from the RNA source)can kill the reaction. Other possible inhibitors of the enzyme are highconcentrations of biotin or tRNA that may have come through as a resultof using the streptavidin-agarose column or other columns. Also, sinceagarose has some nasty compounds in it, if the RNA has been eluted fromthe column using extreme conditions such as heat, this also may cause inhibitorsto be released with the RNA. No one in the lab has done careful experimentsto quantify these problems, and much of the results are anecdotal. Thusthe best thing to do is (1) always quantify your RT efficiency and (2)talk to others who have done RT reactions using a sample similar to yours.
It may be advisable to add RNasin to inhibit RNases. However, DO NOT ADD THIS INHIBITOR BEFORE DOING THE HEATING/COOLING STEP, which will denature the inhibitor and release co-purified RNase contaminants. Also, make sure to add the DTT before adding RNasin, since RNasin also requires reducing conditions.
Superscript II procedures from Gibco BRL
Superscript II product description from GibcoBRL
Reverse Transcription: Frequently Asked Questions from Gibco BRL
David Wilson, Jan 1998