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

Abstract

1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia and rapid quantification results (Pfaffl, 2001; Yuan et al., 2006). Because of the lacking consensus on how to best perform qPCR, MIQE guidelines have been developed to uniform qPCR experiment setup, optimization and data analysis, making the protocols comparable between different research groups and organizations and ensuring the relevance, accuracy, correct interpretation and repeatability of the results (Bustin et al., 2009). When analyzing gene expression, qPCR data can be subjected to absolute or relative quantification (Livak and Schmittgen, 2001; Pfaffl, 2001; Yuan et al., 2006). Absolute quantification employs internal or external caliComparison of methods for relative quantification of gene expression using real-time PCR 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.