Real Time Quantitative PCR has become a staple of almost every molecular biology lab in the world. In fact, I doubt that if asked, many scientists would be willing to admit that they don’t do real time quantitative PCR. The cost of attaining a Real Time qPCR instrument and the per assay costs have come down exponentially putting real time quantitative PCR within the reach of most molecular biology labs.
In this video tutorial by Bio-Rad Laboratories (which is applicable to ALL real time quantitative PCR platforms), you will learn the basics of Real Time qPCR and the science behind absolute and relative quantification methods. Absolute quantification is used when looking to answer the question of “how many” or “how much.” Examples include how many copies of gene x are in my sample or how much viral load is there in a given biological sample. Relative quantification, (the more popular technique for data analysis), is used when looking to measure changes or differences in gene expression between various samples. Examples include looking for upregulation or downregulation of a gene in a normal versus disease state or changes in gene expression following an siRNA gene silencing experiment.
Several methods have been published for conducting real time qPCR data analysis using relative quantification methods including Livak, delta CT and the Pfaffl methodology. The video will also explain the differences between each of these techniques and a review of their uses and limitations.
If you are very ambitious, visit the original Bio-Rad video link for a clearer picture that combines both of these youtube videos into one (youtube doesn’t allow for videos that are over 10 minutes long and tends to make text a bit blurry).