Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula

For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative C T (ΔΔC T ) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression’s C T Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.