To test the effect of plasmid DNA MK-0683 HDAC inhibitor conformation on the accuracy of DNA quantification, we used UV absorbance as well as 2 fluorescent dye-binding methods: Hoechst 33258 dye-binding assay and Quant-iTTM dsDNA BR assay. In addition to these methods, which are used in routine molecular biology, a hydrolysis/HPLC DNA analysis method, which is conformation independent, was also included as a reference. In this study, plasmid DNA samples were directly used as DNA standard after preparation to avoid DNA measurement error. The effect of plasmid DNA conformation was then investigated using 4 qPCR chemistries, including SYBR Green, TaqManH, TaqManH MGB, and TaqManH LNA, by preparing DNA standard curves using supercoiled, nicked-circular, closed-circular, and linear plasmids. The quantification bias caused by plasmid standard DNA conformation is of great concern in qPCR applications, which rely heavily on the accuracy of DNA quantification. Thus, the possible mechanism, consequences, and resolution of quantification bias caused by plasmid DNA conformation were investigated in this study. Our results suggest that a detailed protocol of plasmid standard DNA preparation should be followed to ensure the accuracy and reproducibility of DNA quantification and qPCR analysis. Quantitative real-time PCR is a powerful technique that allows direct quantification of absolute copy number of DNA without post-PCR manipulation. There are 3 important variables that need to be taken into consideration when preparing a plasmid DNA calibration curve for qPCR. The first is the conformation of the DNA. It has been reported that PCR is very sensitive to conformational changes in template DNA, especially the change from supercoiled to linear, and that the use of supercoiled plasmid DNA standards caused serious overestimation of microalgae gene copy number. The second variable is the method used to quantify the DNA. The results of this study suggest that the effect of DNA conformations to quantification methods is variable. The final variable is the qPCR method used to analyze the DNA, which is also highly variable depending on the conformation of the DNA. From this, it is clear that the generation of an accurate DNA calibration curve begins with the control of the standard plasmid DNA conformation. The qPCR quantification bias caused by plasmid DNA conformation and its possible mechanism have been reported in previous studies. As the result of this study, closed-circular/ supercoiled plasmids showed significant lower Ct value to nickedcircular/linear plasmids in these report. However, it is difficult to conclude these findings because different DNA measurement methods were used in these studies. Since the same amount of plasmid DNA in supercoiled and other conformations may have different result of DNA measurement, quantification of plasmid DNA samples after enzyme preparation will introduce DNA measurement error into the result of qPCR. Thus the DNA measurement error caused by plasmid DNA conformation may also contribute to the quantification bias in the previous studies. For these reasons, plasmid DNA samples were directly used as DNA standards after preparation to avoid DNA measurement error in this study.