RT-PCR数据分析方法.pdfVIP

METHODS 25, 402–408 (2001) doi:10.1006/meth.2001.1262, available online at on Analysis of Relative Gene Expression Data Using Real- C Time Quantitative PCR and the 2 T Method Kenneth J. Livak* and Thomas D. Schmittgen†,1 *Applied Biosystems, Foster City, California 94404; and †Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534 of the target gene relative to some reference group The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantifica- such as an untreated control or a sample at time zero tion and relative quantification. Absolute quantification deter- in a time-course study. mines the input copy number, usually by relating the PCR signal Absolute quantification should be performed in situ- to a standard curve. Relative quantification relates the PCR signal ations where it is necessary to determine the absolute of the target transcript in a treatment group to that of another transcript copy number. Absolute quantification has sample such as an untreated control. The 2CT method is a been combined with real-time PCR and numerous re- convenient way to analyze the relative changes in gene expression ports have appeared in the literature (6–9) including from real-time quantitative PCR experiments. The purpose of this two articles in this issue (10, 11). In some situations, report is to present the derivation, assumptions, and applications of the 2CT method. In addition, we present the derivation and it may be unnecessary to determine the absolute tran- applications of two variations of the 2CT