Table 2 Effect of protocol optimization and RNase H on qPCR efficiency and linearity
VEGFB | MMP9 | TFRC | HAMP | GAPDH | Mean ± SD | P vs -RNase H | P vs original protocol | P vs original protocol/- RNase H | |
|---|---|---|---|---|---|---|---|---|---|
Efficiency (slope) | Original protocol | ||||||||
- RNase H | -1.50 | NA | -3.59 | -1.15 | -1.59 | -1.96 ± 1.10 | |||
+ RNase H | -3.74 | NA | -3.63 | -3.13 | -3.24 | -3.43 ± 0.30 | 0.003 | ||
Optimized protocol | |||||||||
- RNase H | -1.13 | -1.24 | -2.53 | -1.26 | -0.65 | -1.36 ± 0.70 | 0.13 | ||
+ RNase H | -3.28 | -2.83 | -3.04 | -2.71 | -2.54 | -2.88 ± 0.29 | <0.001 | 0.16 | 0.02 |
Linearity (R 2 ) | Original protocol | ||||||||
- RNase H | 0.66 | NA | 0.97 | 0.63 | 0.95 | 0.80 ± 0.18 | |||
+ RNase H | 0.99 | NA | 0.98 | 0.99 | 0.97 | 0.98 ± 0.01 | 0.16 | ||
Optimized protocol | |||||||||
- RNase H | 0.99 | 0.91 | 0.99 | 0.72 | 0.87 | 0.90 ± 0.11 | 0.38 | ||
+ RNase H | 0.98 | 0.97 | 0.97 | 0.99 | 0.98 | 0.98 ± 0.01 | 0.19 | 0.64 | 0.16 |