Absolute vs. Relative Quantification for qPCR

When we are calculating the results of real-time PCR experiment, there are two major ways to do the quantification, namely absolute or relative quantification. In absolute quantification, you need to use a set of series of diluted PCR products (or plasmids) with known concentrations as a standard to make a standard curve, so that you can quantitate unknowns based on a known quantity. This method is more often used in analyzing correlating viral copy number with a disease state.

However, in relative quantification, you won’t be able to tell the absolute quantity (e.g. 3 copies for gene A) of a certain gene. Instead, you would need to analyze changes in gene expression in a sample relative to another reference sample (e.g. control group) using the expression level of an internal housekeeping gene for calibration.In this case, you would be able to measure the response of a certain gene (or a set of gene) before and after the treatment given.