RCC Cell Line Transfection Protocol

The human ccRCC cell line, 786-O, was obtained from the National Collection of Authenticated Cell Cultures of China (No. TCHu186). The 786-O cells were cultured in culture medium RPMI-1640 (C11875500BT, GIBCO by Life Technologies, USA), supplemented with 10% fetal bovine serum (Hyclone, Life Sciences, Shanghai, China). Cells were incubated in a humidified atmosphere incubator of 5% CO₂ at 37°C. 786-O cells have been transfected with double-stranded siRNA according to the protocol of the manufacturer using plasmid using Lipofectamine 3000 reagent (RiboBio) in a six-well plate, as previously described (17 (link)). The transfection dose for each well was 15 μl of siRNA1 (sequences: GGTGCACCTTCATCAACAA), siRNA2 (sequences: GGATTACCAGCGATTTCTA), or non-targeting siRNA (negative control) at a concentration of 10 nmol/L after incubated using RPMI-1640 for 20 min. 786-O cells were harvested for at least 24 h after transfection for further experimental analysis.