FIG 6.
Inhibition of seasonal IAV replication by pretransfection with a cIAV DsiRNA swarm in A549 cells. Human lung epithelial A549 cells (in 12-well plates; 5 × 105 cells/well) were mock transfected (control or no-siRNA bars) or pretransfected with the indicated control and IAV-specific siRNA/DsiRNAs (10 nM). After 21 h of incubation, cells were infected with the indicated IAVs at an MOI of 1 for an additional 24 h. Cellular or the supernatant samples from A549 cells were then collected for RNA isolation and quantitative RT-PCR analysis. (A) The values of RT-PCR analyses were normalized against β-actin gene-specific mRNA, and the relative cellular IAV M1 mRNA level was calculated by the ΔΔCT method using untreated cells as a calibrator. The means (±SD) of three parallel analyses are shown. Data are representative of three individual experiments. Statistical significance was determined against results from samples of nontransfected cells (boxed bars). *, P < 0.05. (B) Cell culture supernatant viral RNA levels from isolated total RNA were calculated relative to the level in untreated control cell supernatants with the ΔΔCT method.