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RT-PCR - (Nov/20/2011 )


This may be a very dumb question, but I haven't done rt-pcr before...

So, I'm doing a reverse transcription on my RNA. I have designed 5 rt primers along the length of the RNA.

My question is: should I put all the primers in one reaction, so that I get a pool of cDNA of different lengths?

Or should I have a separate reaction for each primer?

My goal is to determine the ratio between the long, full-length RNAs Vs. the shorter ones.

Thanks a lot!


I think you should use random primers or oligo dT primers to do the RT reaction first, then use different primers sets to amplify different isoforms of the gene and compare their relative expression.