Does non-digested RNA in cDNA sample have an effect on RT-PCR quantification? - (Feb/16/2007 )
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?
normally when you use kits to reverse transcribe RNA there is an RNAse digestion step in the end.
The primer can also bind RNA.
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.