2 replies to this topic

[cellthetruth]

Hello everybody

i'm doing a qRT-PCR for the first time in my lab-career. My supervisor told me I need to do an RNase H treatment after cDNA synthesis. But I don't understand why!??? The DNA polymerase should'nt be able to amplify my GOI from RNA, that's the reason for cDNA synthesis. Or am I wrong here? Is there any other reason?

Thanks for help / explanations

[jerryshelly1]

Listen to your lab supervisor.  I would suggest, since you are a new researcher, to evaluate and study every new procedure.  This will allow you to understand why each step is necessary for a sufficient end result.  The RNase H is added to remove any remaining RNA that is present following RT.  If the RNA were left in the solution, it may form an RNA:DNA hybrid that could potentially cause inhibition during DNA synthesis.  This is important if your cDNA product is of short length, which would greatly hinder proper DNA pol binding and correct replication.  RT-PCR is very sensitive and any inhibition in DNA replication would drastically affect your Ct values.

Edited by jerryshelly1, 26 February 2013 - 10:55 AM.

[cellthetruth]

Thank you for the comprehensible explanations.