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Does non-digested RNA in cDNA sample have an effect on RT-PCR quantification? - (Feb/16/2007 )

Hello everyone!

I was thinking about something. Suppose that you reverse transcribed your total RNA with an RNase H(-) RT to obtain cDNAs for longer transcripts. Then, if you directly use this cDNA for an RT-PCR reaction, you put into the tube both the cDNA and the total RNA.

What I want to know is, is it possible for PCR primers to anneal to transcripts at 58 C? If then, what I amplify is both the RNA and the cDNA, right?

In that case, should I digest the RNA after the RT reaction?

-jahan-

hi,

normally when you use kits to reverse transcribe RNA there is an RNAse digestion step in the end.
The primer can also bind RNA.
Cheers

-Bomber-

Amplification of some PCR targets (>1 kb) may require the removal of RNA complementary to the cDNA.

To remove RNA complementary to the cDNA, add 1 µl (2 units) of E. coli RNase H and incubate at 37°C for 20 min.

-pcrman-