用于比较定量PCR分析的误差最小化算法：Q-Anal。

OConnor William, Runquist Elizabeth A

Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA.

Anal Biochem. 2008 Jul 1;378(1):96-8. doi: 10.1016/j.ab.2008.03.041. Epub 2008 Mar 28.

Current methods for comparative quantitative polymerase chain reaction (qPCR) analysis, the threshold and extrapolation methods, either make assumptions about PCR efficiency that require an arbitrary threshold selection process or extrapolate to estimate relative levels of messenger RNA (mRNA) transcripts. Here we describe an algorithm, Q-Anal, that blends elements from current methods to by-pass assumptions regarding PCR efficiency and improve the threshold selection process to minimize error in comparative qPCR analysis. This algorithm uses iterative linear regression to identify the exponential phase for both target and reference amplicons and then selects, by minimizing linear regression error, a fluorescence threshold where efficiencies for both amplicons have been defined. From this defined fluorescence threshold, cycle time (Ct) and the error for both amplicons are calculated and used to determine the expression ratio. Ratios in complementary DNA (cDNA) dilution assays from qPCR data were analyzed by the Q-Anal method and compared with the threshold method and an extrapolation method. Dilution ratios determined by the Q-Anal and threshold methods were 86 to 118% of the expected cDNA ratios, but relative errors for the Q-Anal method were 4 to 10% in comparison with 4 to 34% for the threshold method. In contrast, ratios determined by an extrapolation method were 32 to 242% of the expected cDNA ratios, with relative errors of 67 to 193%. Q-Anal will be a valuable and quick method for minimizing error in comparative qPCR analysis.

目前用于比较定量聚合酶链反应（qPCR）分析的方法，即阈值法和外推法，要么对PCR效率做出需要任意选择阈值过程的假设，要么进行外推以估计信使核糖核酸（mRNA）转录本的相对水平。在此，我们描述了一种算法Q-Anal，它融合了现有方法的要素，以绕过关于PCR效率的假设，并改进阈值选择过程，以将比较qPCR分析中的误差降至最低。该算法使用迭代线性回归来确定目标扩增子和参照扩增子的指数期，然后通过最小化线性回归误差来选择一个荧光阈值，此时两个扩增子的效率均已确定。根据这个确定的荧光阈值，计算两个扩增子的循环时间（Ct）和误差，并用于确定表达比率。通过Q-Anal方法分析了来自qPCR数据的互补DNA（cDNA）稀释测定中的比率，并与阈值法和外推法进行了比较。由Q-Anal法和阈值法确定的稀释比率为预期cDNA比率的86%至118%，但Q-Anal法的相对误差为4%至10%，而阈值法的相对误差为4%至34%。相比之下，由外推法确定的比率为预期cDNA比率的32%至242%，相对误差为67%至193%。Q-Anal将是一种在比较qPCR分析中最小化误差的有价值且快速的方法。