DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.
DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.
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DOI：
10.1007/s00216-015-8458-z
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发表时间：
2015-03
影响因子：
4.3
通讯作者：
Emslie, Kerry
中科院分区：
文献类型：
--
作者：
Corbisier, Philippe;Pinheiro, Leonardo;Mazoua, Stephane;Kortekaas, Anne-Marie;Chung, Pui Yan Jenny;Gerganova, Tsvetelina;Roebben, Gert;Emons, Hendrik;Emslie, Kerry
6个标准物质(ERM-AD623a-f)的特性赋值已通过使用QX100系统(μ-Rad)的液滴数字聚合酶链式反应(DdPCR)得到验证，每个标准物质含有一个每™100 000到10个拷贝的质粒DNA溶液。用数字聚合酶链式反应测量拷贝数浓度的关键因素之一是分配体积。因此，我们通过光学显微镜测定了平均液滴体积，显示出平均液滴体积比QuantaSoft软件1.3.2.0版(Bio-Rad)中用作计算拷贝数浓度的定义参数的液滴体积小8%。这一观察解释了为什么用ddPCR估计的拷贝数浓度和使用QuantaSoft软件中预定义的平均液滴体积估计的拷贝数浓度系统性地低于dPCR测量的拷贝数浓度，从而在这两种技术获得的值之间造成显著偏差。当用测得的0.834 nL的液滴体积来估计拷贝数浓度时，差异不再显著。新版本的QuantaSoft软件(1.6.6.0320版)已随Bio-Rad的新QX200系统和QX100升级一起发布，它使用0.85nL的液滴体积作为定义的参数来计算拷贝数浓度。在光学显微镜下观察到的由液滴发生器产生的液滴的单分子层
The value assignment for properties of six certified reference materials (ERM-AD623a–f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad’s new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration. Monolayer of droplets generated by the droplet generator and observed under an optical microscope
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