Efficiency in qPCR

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chiharumeow
chiharumeow's picture
Efficiency in qPCR

Could anybody tell me how to improve high Efficiency ?  Sample of samples resulted in Efficiency over 2.0.  I understand that it's caused by primer dimers or unspecific amplification.  The primers were tested with other samples and they produced good dataon different cDNA templates in the sample run. I appreciate if someone give me ideas of how to improve my efficiencies.  Thank you !!

Brad Best
Brad Best's picture
Did you make any progress on

Did you make any progress on this?  I'm guessing that by "sample of samples" you mean a dilution series?  If you've tested your assay with dilution series using other samples and had an efficiency around 90-110%, it would argue against problems with the primers.

Have you done a melt curve?  Nice, single peak?

You could take the end products of your high-efficiency reaction and run them on an agarose gel to see if you're getting more than one product.

If everything else looks good and your primer set works with other samples, I'd guess there's something amiss with your trouble sample.  Perhaps the cDNA isn't pure enough.  High efficiencies can result from PCR inhibitors that increase the Ct at higher concentrations but dilute out at the lower concentrations, resulting in sample Cts being closer than they should be.