oligo(dT) plus random primers in RT - (Jul/20/2006 )

Hallo,

I am using for my cDNA synthesis the SuperScript II reverse transcriptase (Invitrogen). I would like to prime using an association of both oligo(dT) and random primers.
Has someone experience on that ? I know that they should prime at different temperature, so a protocol would be appreciated.

Thanks for any help,

Simone

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you can not use the mixture of oligodT and random primer because for oligo dT transcription is carried out at 42°C but for random primer R-Transcription is carried out at 37°C so you can not use the two oligo in the same time

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I've done it at 42 with random hexamers. But I incubated at 25 for 10 min first. I don't see why you want to reverse transcribe using both

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I want to do it becuase my target gene is towards the 5'UTR of a long mRNA, while the reference gene towards the 3'UTR. Moreover, I am working with samples that show signs of degradation because they have not been supposed to be used for RNA extraction and random priming can partially compensate for this issue.
Simone

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I would simply give it a try. Otherwise, you could reverse transcribe in two different reactions (one with oligo-dT and the other one with random primers) and then mix the two reactions.

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