Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.
Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.
复制标题
DOI：
10.1371/journal.pone.0029101
复制
发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Pan TM
中科院分区：
文献类型：
--
作者：
Lin CH;Chen YC;Pan TM
实时定量 PCR (qPCR) 是特定核酸序列定量的金标准。然而，最近的一份报告揭示了一个严重的问题：超螺旋质粒标准品导致 qPCR 定量的严重高估。在本研究中，我们研究了质粒 DNA 构象对 DNA 定量和 qPCR 效率的影响。我们的结果表明，质粒 DNA 构象对 qPCR 绝对定量的准确性有显着影响。具有不同 DNA 构象的质粒标准品之间的 DNA 标准曲线发生显着变化。此外，DNA测量方法和质粒DNA构象的选择也可能影响DNA标准曲线的测量误差。由于质粒 DNA 构象对 qPCR 准确性的多重影响，应努力确保 qPCR 质粒标准品的最高一致性。因此，我们建议标准质粒 DNA 的构象、制备、定量、纯化、处理和储存应在定量实时 PCR 实验出版的最低信息 (MIQE) 中进行描述和定义，以确保 qPCR 绝对定量的再现性和准确性。
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.
登录
查看更多内容
影响因子：
2.9
作者：
LABARCA, C;PAIGEN, K
通讯作者：
PAIGEN, K
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
3.5
作者：
MATASSI, G;MELIS, R;BERNARDI, G
通讯作者：
BERNARDI, G
DNA超螺旋抑制了实时PCR：一种定量线粒体DNA损伤和修复的新方法。
影响因子：
14.9
作者：
Chen J;Kadlubar FF;Chen JZ
通讯作者：
Chen JZ
影响因子：
4.1
作者：
Shimelis, Olga;Giese, Roger W.
通讯作者：
Giese, Roger W.