RNA interference efficiency. (A) Expression levels were measured by RT-PCR before (Ctr) and 6 days after the respective dsRNA (RNAi) injection based on agglutinin, HSC70B, and EF-1α and β-galactosidase (dsβ-gal) as a control. Primers for RT-PCR were designed from agglutinin, HSC70B, and EF-1α as well as ribosomal protein gene S7 (RpS7). The expression of RpS7 (23 cycles) served as a loading control. (B) The ribosomal protein gene S4 (RpS4) and HSC70B transcript levels (mean ± SD) were measured by quantitative RT-PCR at 6 days after ONNV-eGFP and dsHSC70B and dsβ-gal injections with 3 biological replicates. Primers for qRT-PCR were designed from RpS4 and HSC70B (Table 1). The transcript levels of the loading control (RpS4) did not show significant differences between dsHSC70B and dsβ-gal treatments. However, the HSC70B transcript level in An. gambiae with dsHSC70B injection show an average 58% reduction of transcript levels compared to that of the control mosquitoes with dsβ-gal treatment (Student's paired t-test, P = 0.0047).