All the different sizes of oligo dT seem to have really low Tm (32-39 C) and I've been running my RT reactions at 50 degrees which is around 15 degrees above their Tm values. Is the length of the oligomer going to matter for the efficiency of converting all the mRNA into cDNA? Thanks in advance for your replies.
Instructions for most commercial reverse transcription kits recommend a two stage temperature profile for the enzyme incubation. Usually something like 5 to 10 minutes at 20 to 25 C followed by the higher temperature 37 or 45 or 50 for 30 minutes plus. The purpose of the low temp incubation phase is to allow the short random oligomers or oligo dT to bind and for the Reverse Transcriptase to extend sufficiently from these primed sites to create a structure that will be stable at the higher temperature, where the enzyme is more active and can rapidly produce full length transcripts.
The length of the primer also affects the binding affinity of reverse transcriptase for the primer/template complex. For instance HIV-1 RT prefers primer that are 18 nucleotides in length or longer.
I think Roland's reply gets to the heart of matter and I think is the only real difference you need to worry about so long as you are only want to reverse transcribe mRNA. If you are going after other RNA species, you will want to switch to random hexamers instead of oligo dT. Other than that, the only thing I can think of is if the mRNA you are interested in has an abnormal poly-A tail that is somehow affecting your oligo dT primer annealing. Best of luck.
I agree with the guys above but in my experience with human RNA samples, 12 to 18 nucleotides in length are ok). However, I would suggest using a mixture of random hexamers and oligo-dTs. I get a 1-2 increase in CT value compared to oligos alone. Maybe do a quick experiment to compare oligos only and RH+oligos
Instructions for most commercial reverse transcription kits recommend a two stage temperature profile for the enzyme incubation. Usually something like 5 to 10 minutes at 20 to 25 C followed by the higher temperature 37 or 45 or 50 for 30 minutes plus. The purpose of the low temp incubation phase is to allow the short random oligomers or oligo dT to bind and for the Reverse Transcriptase to extend sufficiently from these primed sites to create a structure that will be stable at the higher temperature, where the enzyme is more active and can rapidly produce full length transcripts.
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