At work, quantification of differential gene expression is undertaken using real time RT-PCR. The technology generates a quantitative end point threshold (Ct) value defined as the threshold cycle where the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold. The threshold is usually placed in the exponential phase of the PCR amplification cycle and is inversely related to the quantity of the amplicon being amplified. The following guidelines review some basic concepts of qPCR data analysis using delta delta Ct with discussions on selecting housekeeping gene and replicate analysis and data reporting. The post is intended for those users who are already familiar with the technology and have a working knowledge of the basics of qPCR.
