Analysis of Relative Gene Expression Data Using Real-

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.

E transcriptase, cDNA was synthesized from 1 mg total RNA isolated X 5 E R 5 E, from human Raji cells. Serial dilutions of cDNA were amplified by real-time PCR using gene-specific primers. The most concentrated sample contained cDNA derived from 1 ng of total RNA. The DC T (C T,c 2myc 2 C T,GAPDH) was calculated for each cDNA dilution. The data

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