Amplification Efficiency of TaqMan Gene Expression Assays

Applied Biosystems has designed and manufactured over 40,000 real-time PCR assays for measuring the expression of genes in humans, mice, and rats. TaqMan Gene Expression Assays each consist of amplification primers (forward and reverse) and a fluorescent labeled TaqMan probe, formulated into a single tube. One of the major user concerns regarding any real-time PCR-based assay product (primers and TaqMan probe) is the amplification efficiency of the PCR reaction. Applied Biosystems has done extensive R&D validation of our TaqMan Gene Expression Assays to address this concern. Two types of experiments have been performed to evaluate the amplification efficiency of TaqMan Gene Expression Assays. First, we determined the amplification efficiency of a large sampling of assays using statistically relevant methods. Second, we evaluated the effect of amplification efficiency on the ability to determine a difference in the relative quantity of a transcript expressed in two different samples. Our results indicate that the amplification efficiency associated with assays designed using our informatics pipeline is 100%. Additionally, our results show that the nnCT method for relative quantitation is better correlated with expected fold change in gene expression than methods using predetermined amplification efficiency values for individual assays. Introduction A key attribute of any real-time PCR reaction is the amplification efficiency of the PCR. Specifically, is there a doubling of PCR product at every PCR cycle? The equation that describes the exponential amplification of PCR is: