Figure 3

In vitro RNAi in tick synganglia. Adult female I. scapularis ticks were dissected and synganglia pooled in two groups and incubated with buffer alone or with (A) dsRNA-β-actin or (B) dsRNA Na⁺-K⁺-ATPase for 6 hrs at room temperature. Total RNA was extracted and cDNA produced. RT-PCR was conducted followed by gel electrophoresis. The I. scapularis Cyclophilin A or G was used as a normalizing factor (lanes 6–7). A) β-actin transcript level demonstrating the efficiency of RNAi in tick synganglia. (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene disrupted samples amplified with β-actin gene specific primers, 4–5: mock and gene silenced samples amplified with Na⁺-K⁺-ATPase gene specific primers, 6–7: mock and gene silenced amplified Cyclophilin A gene specific primers). B) Na⁺-K⁺-ATPase transcription demonstrated the efficiency of RNAi. (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene suppressed samples amplified with Na⁺-K⁺-ATPase gene specific primers, 4–5: mock and gene silenced samples amplified with β-actin gene specific primers, 6–7: mock and gene silenced samples amplified Cyclophilin G gene specific primers). Low DNA Mass™ ladder (100–2000 bp; 2000 bp, 1200 bp, 800 bp, 400 bp, 200 bp, 100 bp) was used from Invitrogen.