Hi!
I want to analyze my taqman assay data by the comparative CT method (delta-delta Ct). We use Rotorgene 6000 and REST 2009 software in our lab. Should I perform validation experiment (to confirm the similar efficiency for primers) prior to analysis or, as it is claimed in the equipment manual, the "Amplification Plot based Efficiency calculation" ability, make it unnecessary.
Hi,
Amplification plot based PCR efficiency calculation makes it redundant to validate PCR efficiency by conventional way, through the serial dilution of template. But, one would not be able to infer the presence of inhibitors in the template if qPCR by serial dilution is not preformed. So, it might be better to do that even if not for PCR efficiency calculation.
I am looking forward to further comments from others.
Hi everybody
It is suggested that efficiency of target gene and house keeping gene should calculate separately by amplification of serial dilution of each gene at least in duplicate or triplicates during an intra assay.
Then consider the correct limits for calculated efficiencies.
Best
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