What is your Protocol for RNAi on Cell Cultures

6-Well Plates Bathing Prepare dsRNA suspended in water. We use ~500 bp dsRNA. Add ~10-30 µg dsRNA to wells of 6-well tissue culture plate. We use 0.1-0.3 µg in 384-well plates for 25-50 nM final concentration. Count cells, then spin to pellet (~1200 rpm, 5'). Resuspend cells at 1-5 x 106 cells/ml in serum free media. Plate 1 ml cells into wells of 6-well plate. It doesn't seem to matter if dsRNA or cells are added first. Incubate dsRNA with cells at RT for 30'. Add 3 ml complete media with 10% FBS to each well. Incubate 3 days and analyze. Length of incubation may vary depending on assay.

384-Well Plates Bathing Remove 384-well plates pre-aliquoted with dsRNA from freezer to thaw. The 384-well plates contain 5ul of ~0.05ug/ul dsRNA in water for ~0.25ug dsRNA/well. The dsRNAs are ~500 bp. Spin plates at ~1200 rpm for 1'. before removing seals. Count cells, then spin to pellet (~1200 rpm, 5'). Resuspend cells at 1-5 x 106 cells/ml in serum free media. Plate 10 ul cells into wells of 384-well plate. Incubate dsRNA with cells at RT for 30'. Add 30 ul of complete media to each well. Seal the plates to prevent evaporation. Incubate 3 days and analyze. Length of incubation may vary depending on assay. Transfection Remove 384-well plates from freezer to thaw. The 384-well plates contain 5ul of ~0.016ug/ul dsRNA in water for ~0.08ug dsRNA/well. The dsRNAs are ~500 bp. Spin plates at ~1200 rpm for 1'. before removing seals.