Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay

• Published in: PloS one Vol. 6; no. 12; p. e29101
• Main Authors: Lin, Chih-Hui, Chen, Yu-Chieh, Pan, Tzu-Ming
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.12.2011; Public Library of Science (PLoS)
• Subjects: Accuracy; Agriculture; Bias; Biological Assay - methods; Biology; Calibration; Chemistry; Chromatography; Cloning; Conformation; Deoxyribonucleic acid; DNA; DNA - analysis; DNA - chemistry; DNA methylation; DNA, Superhelical - genetics; E coli; Error analysis; Escherichia coli; Genetic testing; Genetically modified organisms; Genomes; Laboratories; Life sciences; Medicine; Methods; Molecular biology; Nucleic Acid Conformation; Organic Chemicals - metabolism; Plants, Genetically Modified; Plasmids; Plasmids - analysis; Plasmids - chemistry; Polymerase chain reaction; Purification; Real time; Real-Time Polymerase Chain Reaction - methods; Reference materials; Reproducibility; Spectrum analysis; Trends; Zea mays - genetics; Taiwan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0029101

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. 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. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.