Comparison of methods for relative quantification of gene ex- pression using real-time PCR

Abstract

Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2^−ΔΔCq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2^−ΔΔCq method.