Analysis of Relative Gene Expression Data Using …

METHODS 25, 402 408 (2001). , available online at on Analysis of Relative gene Expression data Using real - time Quantitative PCR and the 2 CT 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.

ANALYSIS OF REAL-TIME PCR DATA 403 1. THE 22DDCT METHOD or X N 3 (1 1 E)DCT 5 K, [6] 1.1. Derivation of the 22DDCT Method The equation that describes the exponential amplifi- where X N is equal to the normalized amount of target cation of PCR is (X0/R 0) and DC T is equal to the difference in threshold cycles for target and …

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