4.13. SiRNA Transfection

FlexiTube siRNA targeting VCAN (Qiagen ID: SI04948587; target sequence CAT GCG CTA CAT AAA GTC AAA) and AllStars Negative Control siRNA were purchased from Qiagen. Briefly, cells were cultured the day before transfection to 60–70% confluence. The siRNA and control were diluted using OptiMEM (Gibco, 11058021) prior to adding the transfection reagent Hiperfect (Qiagen, ID: 301707). After incubation for 5 min, the transfection mixture was added to the wells containing complete medium. A final siRNA concentration of 2.5 × 10⁻⁹ mol∙L⁻¹ and a final ratio of 1.2 µL Hiperfect per 10⁻⁹ mol of siRNA were used. For RNA isolation, cells were harvested two days after transfection. For Transwell membrane migration assays, cells were starved for one day after transfection and, after an additional day, seeded for migration. For the cell proliferation assay, starvation medium was used for transfection and then replaced with complete medium after one day. Data were collected after one additional day.