Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution
Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution
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DOI：
10.1016/j.ymeth.2010.01.003
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发表时间：
2010-04-01
期刊：
影响因子：
4.8
通讯作者：
Livak, Kenneth J.
中科院分区：
文献类型：
--
作者：
Weaver, Suzanne;Dube, Simant;Livak, Kenneth J.
本文以拷贝数变异（CNV）为范例，评估了qPCR的定量分辨率。为实时qPCR数据开发了误差模型，显示CNV测定的精密度如何随重复次数而变化。使用具有不同数量的X染色体的样品，实验数据表明，实时qPCR可以很容易地区分四个拷贝和五个拷贝，这对应于相对数量的1.25倍差异。数字PCR被认为是qPCR的替代形式。对于数字PCR，示出了将CNV测定的精度与反应室的数量相关的误差模型。数字PCR的定量能力与区分四个和五个拷贝的人类基因MRGPRX 1的实验说明。对于实时qPCR或数字PCR，实际应用这些模型以实现增强的定量分辨率需要使用可以同时进行数千个反应的高通量PCR平台。比较这两种方法，实时qPCR具有通量的优势，而数字PCR在数据分析的假设方面具有简单的优势。(C)2010年爱思唯尔公司All rights reserved.
This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. (C) 2010 Elsevier Inc. All rights reserved.