Standard curve

The efficiency of the PCR reaction is very important for correct interpretation of the qPCR results. It is dependent on the assay, the master mix performance, and sample quality. It is calculated based on a standard curve.

A standard curve is made from several qPCR reactions, on a 10X dilution series of DNA. The dilution series can be made from a pool of the cDNA samples you are going to test, successively diluted 10 times. To construct a standard curve you need to plot the Ct-values obtained for each dilution (upper part of figure below) against the logarithm of the dilution factors (DF) used to make the standard curve. The standard curve is then the regression line through these points (lower part in figure below, left side). The slope of the regression line is related to the amplification efficiency by the formula in the figure below. Generally, efficiency between 80 and 110% is considered acceptable. At 100% you are doubling the amount of product in each cycle of your PCR reaction. You can also use the regression line to determine the concentration of unknown samples from their Ct-values.