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Question regarding about PCR - DNA or RNA as template

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Question regarding about PCR - DNA or RNA as template

Postby Xenotoxic » Fri Aug 17, 2012 3:55 am

Hello to all biologists.

I have a question regarding about PCR.

We all know and seems to be common to think of using cDNA as template for PCR. Then my junior had asked me very fundamental question and I could not accurately answer to this question that could solve problem

His questions is following - why DNA must be used for PCR and not RNA???

The question arised from following - I did an experiment which isolated RNA from liver of experimental mice and made cDNA from that RNA. Problem is that I did PCR with RNA and cDNA simultaneously and did gel electrophroesis.

I obtained very interesting results which corresponding bands of housekeeping gene were detected when RNA was used as template but not the target gene. We thought of possibly of genomic DNA contamination but when I designed primer, I intercalated intron between exon so the band corresponding to genomic DNA should be very large but the band appeared exactly same as its target size.

I told junior that you cannot use RNA as template cause 1. RNA will degrade during 95 C denaturation step or 2. DNA polymerase used for PCR is very specific for DNA.

Do you guys have any other answers?

Thank you.
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Postby FlyMusings » Sat Sep 29, 2012 5:45 pm

Do you have controls with the RNA without PCR, run on a gel? I am also a little surprised that PCR can amplify RNA.
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