Resolution:
## Figure 5.
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 (R^{2}) corresponds to the correlation coefficient of the regression line. A coefficient
R^{2 }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 R^{2 }= 1. E was improved by RNase H treatment.
Jeanty |