Verifying RNAi Efficacy via qPCR

To verify the effectiveness of RNAi, approximately 200 female adults of SS, AbR, FeR, and CyR strains, after feeding with dsRNA for 48 hours, were collected for each sample, and were replicated for three times. The primers of qPCR for TCE2 gene were designed by using primer 3.0 (http://frodo.wi.mit.edu/)55 . RPS18 (FJ608659) and α-TUB (FJ526336) were used as stable reference genes (Supplementary Material: Table S1)56 (link). The qPCR was performed on an Mx3000P thermal cycler (Agilent Technologies, Inc., Wilmington, NC, USA) with 20 μL reaction mixtures containing 1 μL cDNA, 10 μL iQ™ SYBR® Green Supermix, 1 μL of each gene-specific primer (0.2 mM) and 7 μL ddH₂O. The optimized qPCR protocol used for amplification was: 95 °C for 2 min, then 40 cycles of denaturation at 95 °C for 15 s, 60 °C for 30 s and elongation at 72 °C for 30 s. Finally, melt curve analyses (from 60 to 95 °C) were included at the end to ensure the consistency of the amplified products. The quantification of expression level was analyzed using the 2^−ΔΔCt method57 (link). Differences in expression levels were analyzed by independent-sample t-test with a p-value < 0.05 in SPSS 19.0 (SPSS Inc., Chicago, USA).