RT PCR - dilution prior to PCR (Sep/02/2004 )
I wonder could you answer a basic question for me: Having performed 1st strand synthesis do I have to dilute the cDNA prior to PCR analysis. I designed primers and am using PCR to amplify the product before electrophoresis but I am confused as to whether I need to dilute the cDNA prior to performing the PCR reation. In the promega protocol it says to dilute to 100ul but in others it doesn't mention it.
If your target is a rare message, I would not dilute the cDNA before PCR, however if it is really abundant...diluting would be better for the PCR reaction. If you have enough sample I would try both ways and see which one gives you the best yield.
I didn't do dilution until I found the protocol coming with the promega kit, then I think diluting RT products is good in two ways: giving you more templates and making detecting subtle expression changes easier.