According to the manufacturer information, the QIAprep miniprep kit used in this study also results in mostly supercoiled plasmid. If nicks are introduced at opposite positions on both plasmid DNA strands, e.g., by restriction enzyme digestion, a plasmid is linearized and the supercoiling is relaxed. There is evidence that PCR is suppressed by supercoiling of the template DNA, and that the relaxing of DNA supercoil structure could increase the Betulin efficiency for primer binding and elongation in a PCR reaction. This explains well the higher Ct values for circular plasmid than that for linearized plasmid. However, by multiple linear regression analyses, we did not find efficiency differences between the circular and linear DNA in all qPCR that can account for the differential Ct values. Only in one case did we observe a small difference in efficiency, which however contradicted rather than accounted for the Ct difference. It seems likely that the difference in Ct values and quantification accuracies lie in the first several cycles of qPCR when the supercoiled plasmid is the dominant template. Previous research has shown that the efficiency difference in the first few cycles would result in dramatic different qPCR results, such as DCt measured in this case. However, the efficiencies calculated from the standard Pyridone 6 curves do not reflect the differences in the early amplification stage, because the standard curves were constructed based on the Ct values identified in the exponential amplification stage when linear PCR amplicon has become dominant and quantitatively outcompletes the supercoiled plasmid for amplification. Even if the amplification efficiency were calculated using such other methods as one using fluorescent data collected during PCR, the initial lower efficiency of the supercoiled plasmid DNA still may not be easily detected. While qPCR results from undigested plasmid DNA standard are strikingly different from those based on linear standards, linearized plasmid and linear PCR amplicon provide similar quantifications. This suggests that the length and source of the DNA template does not have significant effect on PCR efficiency.