RNAi suppression assays were performed in both Aag2 and S2 cells. Aag2 cells (1.7 × 105 per well in a 24-well plate) were infected with RVFV MP12 or the positive-control CYV at an MOI of 5 and incubated for 24 h at 28°C. Cells were then cotransfected with 100 ng pIZ-Fluc (64 (link), 65 (link)), 40 ng pAct-Renilla (66 (link)) (FLuc and RLuc expression plasmids, respectively), and 0.05 ng dsRNA targeting FLuc or a control dsRNA targeting eGFP using Lipofectamine 2000. At 24 h p.t., cells were lysed in passive lysis buffer and FLuc and RLuc expression was determined using Bright-Glo and Renilla-Glo substrates (Promega).
RNAi suppression assays in S2 cells have previously been described (67 (link), 68 (link)). Briefly, 5 × 104 S2 cells were seeded in 96-well plates and left to adhere overnight. Cells were transfected with 50 ng pMT-Fluc and 15 ng pMT-Rluc for 24 h at 28°C, after which the cells were either infected with RVFV MP12 at an MOI of 5 for 24 h or left uninfected. A 25-ng volume of dsRNA targeting either FLuc or eGFP as a control was fed to the cells. At 7 h after the feeding of dsRNA, expression of luciferase reporters was induced by addition of 0.5 mM CuSO4 per well. After 24 h, cells were lysed and luciferase expression was determined as described above.
RNAi suppression assays in S2 cells have previously been described (67 (link), 68 (link)). Briefly, 5 × 104 S2 cells were seeded in 96-well plates and left to adhere overnight. Cells were transfected with 50 ng pMT-Fluc and 15 ng pMT-Rluc for 24 h at 28°C, after which the cells were either infected with RVFV MP12 at an MOI of 5 for 24 h or left uninfected. A 25-ng volume of dsRNA targeting either FLuc or eGFP as a control was fed to the cells. At 7 h after the feeding of dsRNA, expression of luciferase reporters was induced by addition of 0.5 mM CuSO4 per well. After 24 h, cells were lysed and luciferase expression was determined as described above.
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