re-amplification in real-time PCR - (May/21/2009 )
Does anyone have an experience in preamplification of samples before running real-time PCR? Repeatedly, I don't get enough cDNA for efficient real-time PCR amplification. Would it be possible to amplify in regular PCR for some 30 cycles and than to use the resulting solution as a template for real-time PCR? Also, would it be worthy to try to amplify in more than 40 cycles of real-time PCR? What are the limitations? I'm not experienced in molecular biology, so if I make some obvious mistake in the reasoning, please don't hesitate to explain me the basics.
It is not a bad idea to preamplify before real-time PCR. As a rule of thumb, I don't use more than 36 cycles for RT-PCR. Any amplification after 36 cycles is not real. If you have enough RNA, the best thing to do is to generate enough cDNA by RT reaction.