a half less of plasmid copy number in the solution resulting from ddPCR than qPC - (Aug/16/2016 )
I am try to compare two methods to calculate copy number.
I diluted plasmid in serial concentrations just like doing standard curve.
and then used the same amount of diluted plasmids to do qPCR and ddPCR.
They are supposed to give me the same results of plasmid copy number.
However, the data from ddPCR was always a half less than that of qPCR and the real copy number calculated based on plasmid concentration.
I have tested the amplification efficiency of primers and it is over 90%. So the primers are good.
What else should I check?
Could anyone give me some suggestion why I always got non-consistent between ddPCR and qPCR?
Perhaps this article could shed some insight?
How are you calculating plasmid concentration, what is the conformation of your plasmid, and could that conformation change somehow in the ddPCR reaction versus the qPCR reaction?
Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay
"However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR."
They also show that plasmid conformation can cause significant variation in plasmid quantification by UV, Hoechst 33258, and Quant-iT BR, with the Hoechst assay showing the least variation.