A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res-英文文献.pdf

© 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 9 00 A new mathematical model for relative quantification in real-time RT–PCR Michael W. Pfaffl* Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Germany Received December 18, 2000; Revised February 21, 2001; Accepted March 14, 2001 ABSTRACT fication types in real-time RT-PCR are possible. (i) A relative quantification based on the relative expression of a target gene Use of the real-time polymerase chain reaction (PCR) versus a reference gene. To investigate the physiological to amplify cDNA products reverse transcribed from changes in gene expression, the relative expression ratio is mRNA is on the way to becoming a routine tool in adequate for the most purposes. (ii) An absolute quantification, molecular biology to study low abundance gene based either on an internal or an external calibration curve expression. Real-time PCR is easy to perform, (1,3). Using such a calibration curve, the methodology has to provides the necessary accuracy and produces reli- be highly validated and the identical LightCycler PCR amplifi- able as well as rapid quantification results. But accu- cation efficiencies for standard material and target cDNA must rate quantification of nucleic acids requires a be confirmed (4–6). Nevertheless, the generation of stable and reproducible methodology and an adequate mathe- reliable standard material, either recombinant