Flextube sirna

siRNA treatment procedure, cell viability, and virus titer determination are described in detail in Watanabe et al. (32 (link)). Briefly, two siRNAs per candidate gene were selected from a predesigned genome-wide human siRNA library (FlexTube siRNA; Qiagen). AllStars Negative Control siRNA (Qiagen) was used as a negative control. The siRNA against the NP gene of WSN virus (GGA UCU UAU UUC UUC GGA GUU) purchased from Sigma-Aldrich was used as a positive control. HEK293 cells were transfected twice with 25 nM (final concentration, 50 nM) siRNA duplexes using RNAiMAX (Invitrogen). At 24 h after the second transfection, cell viability was determined using the CellTiter-Glo assay system (Promega) following the manufacturer’s instructions. To assess influenza virus replication, two parallel sets of siRNA-transfected cells were infected with 50 plaque-forming units (PFU) of WSN virus per well of a 24-well tissue culture plate at 24 h after the second siRNA transfection. At 48 h postinfection, supernatants were harvested, and virus titers were determined by plaque assay in MDCK cells.

Cells were plated in 6-well culture plates in 3 ml of RMPI-1640 media containing 10% FBS, and transfected for 48 hours, using FlexTube siRNA (Qiagen, Valencia, CA) targeting STAT3-Lipofectamine 2000 (3μl; Invitrogen), as per manufacturer’s instructions with a final concentration of siRNA at 50 mM. A non-targeting siRNA was used as a negative control.