Cell RNAi (6-well)

This protocol is for RNAi screening of Drosophila cells in 6-well plates using the “bathing” method of delivery (no transfection reagent). We also provide a protocol for RNAi screening in 384-well plates using the bathing method or a transfection method for delivery of the RNAi reagents. That protocol includes notes on 96-well plate screening.

  1. Bathing method of RNAi reagent delivery
  2. Prepare dsRNA suspended in water. We use ~500 bp dsRNA.
  3. 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.
  4. Count cells, then spin to pellet (~1200 rpm, 5').
  5. Resuspend cells at 1-5 x 106 cells/ml in serum free media.
  6. Plate 1 ml cells into wells of 6-well plate.
  7. It doesn't seem to matter if dsRNA or cells are added first.
  8. Incubate dsRNA with cells at RT for 30'.
  9. Add 3 ml complete media with 10% FBS to each well.
  10. Incubate 3 or more days and analyze (optimization should include testing different incubation periods).

Length of incubation may vary depending on assay.

Troubleshooting tip #1

The ratio of cells in serum free to dsRNA in water should be 2:1 during the 30 minute serum starvation step.

Troubleshooting tip #2

If you see unexpected cell death after 24 hours incubation, try using complete media with 12% FBS instead of 10% in step 7. This will make your final concentration closer to 10% FBS.