Hi everyone. For my project I have to reduce the time for reverse transcription of mRNA.
We are using High Capacity cDNA Reverse Transcription Kit from Applied Biosystems.
It's recommended there to use the the extension time of 120 minutes.
The same time I see the general recommendation for MMLV RT 60 min.
Where came this 120 min from?
Could anyone suggest where I can read something about it?
Thanks in advance!
It may be that the extra time is not needed to extend the primers but for rare or very low concentration rna the extra time is added to allow the primers to anneal to all of the available rna molecules as the rate of reaction of primer/hexamer binding is dependent on the concentrations of both rna and primer so if the [RNA] is low then so is the speed at which annealing occurs. Sorry this is a guess and I know of no published work to support it.
Laurence Stuart Dawkins-Hall
en Paul RutlandThank you very much! I will try to read about it.
I've just found an experiment of reducing polymerization time for RT (random hexamers) without affect Cq even for 2,251bp UBC mRNA low expression level.
.
https://nld.promega.com/resources/pubhub/2018/effect-of-a-shortened-reverse-transcription-time-on-qpcr-amplification/#summary.%20https://nld.promega.com/resources/pubhub/2018/effect-of-a-shortened-reverse-transcription-time-on-qpcr-amplification/#summary
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