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Dumb Reverse Transcription PCR Question


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#1 dreww

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Posted 09 May 2010 - 12:39 AM

In Reverse Transcription PCR. with the addition of Reverse Transcriptase, are all ssRNA templates reverse transcribed? if not all, are there non targeted sequence that get reverse transcribed?

#2 JackieG

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Posted 09 May 2010 - 10:31 AM

In Reverse Transcription PCR. with the addition of Reverse Transcriptase, are all ssRNA templates reverse transcribed? if not all, are there non targeted sequence that get reverse transcribed?



I suppose this would depend on the primers you are using, gene specific? or random hexamers?

#3 Trof

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Posted 10 May 2010 - 05:44 AM

As JackieG wrote that would depend on primers. There are basically three types of reverse transcription primers. Random hexamers are as stated primers with random nucleotides, that transcribe everything, and are comonly used when you want to test multiple genes on PCR and including the rRNA (like 18S).

Another option is to use oligo dT primers, that transcribe only full length mRNA with poly A tail. You can do PCR for multiple genes then, but not 18S.

Or you can use gene specific primers, same as for PCR to only transcribe your target gene. This is usualy done in one-step RT protocol.

We routinely use 1:1 mix od random hexamers and oligo dT primers to enrich the mRNA fraction.

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