The Use & Usefulness of Amplification Curve Analysis in Quantitative PCR

Gene Target Solutions presents Jan M Ruijter - Dept Anatomy, Embryology & Physiology, Academic Medical Centre, The Netherlands.
Quantitative real-time RT-PCR (qPCR for short) is a well established technique for the quantification of small amounts of RNA. By monitoring the increase in generated DNA, and determining at which cycle (Cq) a pre-set threshold is reached, the starting concentration can be calculated. However, qPCR data analysis results reveal considerable differences in variability and bias, which in turn may lead to loss of sensitivity and opposing biological conclusions. Most current qPCR analysis methods depend on the proper estimation of the Cq value and the PCR efficiency. Often a dilution series is used to determine the efficiency but this procedure has been shown to lead to variable results. Alternatively, this amplification efficiency can be derived from the individual amplification curves. Recently, all publicly available methods to perform such a PCR amplification curve analysis were compared. The results of this comparison will be described and discussed.