Pre-amplification of template for qPCR - (Apr/13/2010 )
I got another question. What is your opinion about template preamplification before qPCR?
Somebody in my lab once used this process to amplify the amount of template in his sample so that he could evaluate efficiency (by the dilution series method) within a wider range of log cycles (testing the linear dynamic range at more concentration points?).
In his experiment he preamplified his template 14 cycles using the same Taq and reagents he used for qPCR. Then he diluted this preamplified products from 10^0 to 10^-9. At the end he got neat amplification curves each spaced about the ideal 3.32 cycles from each other. He also prepared a dilution in which template amount was equivalent to the original not-preamplified template. The amplification plot of this dilution was just 2/10 of a cycle off from that of the not-preamplified template.
I was just thinking this could be useful when the amount of sample is very limited, I have read a couple of papers in which they test (using special kits) pre-amplification of all cDNAs using kits where they report biased pre-amplification as a potential problem. In my friend's experiment, he used the same primers and reagents (except SYBR) he used for his qPCR.
Basically I wanted to know your opinion about this procedure.
Pre-amplification should be ok if they are only used for the standard curves, but make sure that your sample concentration is within the sample curve range. I wouldn't advise doing pre-amps for sample curves.