RNaseH improves qPCR efficiency. Different quantities of AA-aRNA from universal reference RNA (100-1000 ng) were used as inputs into either the original RT protocol or the optimized RT protocol, with or without RNase H treatment. Resulting cDNAs were diluted 10-fold and subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Cq values were plotted against the Log of the concentration of the AA-aRNA used for RT and linear regression was applied. qPCR efficiency (E) was calculated by the slope of the regression line. A slope of -3.2 indicates optimal efficiency. qPCR linearity (R2) corresponds to the correlation coefficient of the regression line. A coefficient R2 of 1 indicates optimal linearity. (A) Representative experiment using VEGFB primers. (B) Plots representing qPCR efficiency as a function of linearity for the 5 genes tested. Optimum conditions are indicated at the intersection of dotted lines corresponding to E = -3.2 and R2 = 1. E was improved by RNase H treatment.
Jeanty et al. BMC Genomics 2010 11:542 doi:10.1186/1471-2164-11-542