Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data
Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data
复制标题
DOI：
10.1016/s0304-3940(02)01423-4
复制
发表时间：
2003-03-13
影响因子：
2.5
通讯作者：
Moorman, AFM
中科院分区：
文献类型：
--
作者：
Ramakers, C;Ruijter, JM;Moorman, AFM
使用荧光染料SYBR绿色1通过监测产物形成的实时聚合酶链反应（PCR）定量mRNA正广泛用于神经科学、发育生物学和医学诊断。大多数PCR数据分析程序假定感兴趣的扩增子的PCR效率是恒定的，或者在比较C方法的情况下，甚至等于2。当PCR效率仅在0.04范围内变化时，后一种方法已经导致4倍误差。扩增子的PCR效率通常由基于已知RNA输入或参考cDNA样品的稀释系列的标准曲线计算。在本文中，我们表明，第一种方法可以导致PCR效率在0.2的范围内变化，而第二种方法可能会关闭0.26。因此，我们建议对每个循环数数据的Log（荧光）进行线性回归，作为计算每个样品的mRNA起始浓度和PCR效率的无干扰方法。如有要求，可提供进行这一计算的计算机程序（电子邮件：bioinfo@amc.uva.nl;主题：LinRegPCR）。(C)2003爱思唯尔科学爱尔兰有限公司保留所有权利。
Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green 1 is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C, method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample. A computer program to perform this calculation is available on request (e-mail: bioinfo@amc.uva.nl; subject: LinRegPCR). (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.