first strand or double strand cDNA before PCR: RT-qPCR - (Jun/24/2005 )

Should we make first strand or double strand cDNA before PCR? Does a reverse transcriptase that has an endo H activity make specifically only double strand cDNA or can it also make first strand cDNA? And should I use endo H activity to digest the RNA strand template before PCR or not?

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Reverse transcription transcribes RNA to cDNA. RNA is single stranded for the most part, so the concept of making double stranded cDNA is odd to me. I guess you are referring to the cDNA:RNA hybrid that is produce during transcription. In this case, typical RT always produce this hybrid which alot people think of as single stranded cDNA.

I recommend digesting the RNA in your template before use even though its not absolutely necessary.

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I am talking about real time PCR. Does your reply mean for it?

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Yes. In PCR with relation to genomic DNA, your primers target both strands of DNA to produce to two products.

In realtime-RT PCR (people use RT to mean realtime which causes confusion since RT has alway been used to describe reverse transcription), only one target is available since the cDNA is representative of single stranded RNA. The second target only becomes available after the first cycle of PCR.

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