The utility of droplet digital PCR in gene quantification/copy number determination is becoming increasingly prevalent in industry. Its immediate benefits over traditional absolute quantitative PCR methods is hard to deny as it obviates the need to either generate rigorous standard curves or measure PCR efficiency of every gene pair to be measured. ddPCR delivers the same sensitivity as with conventional approaches with the added benefit of smaller errors. ddPCR relies on the Poisson algorithm and is essentially an end point measurement where the sample to be measured is randomly distributed into discrete partitions containing either none, one or more nucleic acid template copies. These individual partitions are then thermally cycled as in conventional PCR to their end point and then read to determine the fraction of partitions that are positive for the amplification. Continue reading
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.