Superscript III - No second strand synthesis? - (Oct/04/2010 )
I want to make sure that I have a proper understanding of cDNA synthesis. Superscript III does only give rise to one strand of cDNA per mRNA, right?
Further, if that is the case, what consequences does this have for doing real-time PCR on this template? Does one of your primers then act to generate the second strand? I'm a little confused.
AFAIK all RT enzymes create only one strand, or better to say a strand complementary to mRNA (that's why it's called "complementary DNA"). On their RNase H activity depends if the mRNA strand stays in duplex with the cDNA or if it's degraded, leaving only single-strand cDNA.
But generaly for subsequent qPCR, RT mix should have RNase H activity, because single-stranded cDNA is quantified more accurately than RNA-DNA duplex.
So you only have one cDNA strand in the first cycle of PCR, only one primer anneals creating second strand. Then you go as usual.