Evaluation of MDA5 Overexpression in DF-1 Cells

When DF-1 cells were approximately 90% full in a 24-well plate, and 0.5 μg of Flag-MDA5-1 and Flag-MDA5-2 plasmids were transfected into DF-1 cells using Lipofectamine^® 3000 (L3000015, Thermo Fisher Scientific, Waltham, MA, USA) transfection reagent. At 24 h post-transfection, total cellular RNA was collected with Trizol to detect the expression of β-IFN, MAVS, and PKR genes. Additionally, DF-1 cells transfected with the plasmids for 24 h were inoculated with H9N2 virus (0.1 MOI). At 6 h post-transfection, the cellular RNA was extracted to determine the expression of viral HA, NA, and NS genes, of which the primer sequences are shown in Table 1.