The protocols listed here are for Drosophila cells in 6 well plates and our pre-aliquoted 384 well plates. RNAi experiments may be done in other size plates, just scale up or down.

Adopted from Clemens et al., PNAS 2000 vol 97(12): 6499-6503

1. 6-Well Plates
   1. Bathing
      1. Prepare dsRNA suspended in water.
         We use ~500 bp dsRNA.
      2. 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.
      3. Count cells, then spin to pellet (~1200 rpm, 5').
      4. Resuspend cells at 1-5 x 106 cells/ml in serum free media.
      5. Plate 1 ml cells into wells of 6-well plate.
         It doesn't seem to matter if dsRNA or cells are added first.
      6. Incubate dsRNA with cells at RT for 30'.
      7. Add 3 ml complete media with 10% FBS to each well.
      8. Incubate 3 days and analyze.
         Length of incubation may vary depending on assay.
2. 384-Well Plates
   1. Bathing
      1. 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.
      2. Spin plates at ~1200 rpm for 1'. before removing seals.
      3. Count cells, then spin to pellet (~1200 rpm, 5').
      4. Resuspend cells at 1-5 x 106 cells/ml in serum free media.
      5. Plate 10 ul cells into wells of 384-well plate.
      6. Incubate dsRNA with cells at RT for 30'.
      7. Add 30 ul of complete media to each well.
      8. Seal the plates to prevent evaporation.
      9. Incubate 3 days and analyze.
         Length of incubation may vary depending on assay.
   2. Transfection
      1. 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.
      2. Spin plates at ~1200 rpm for 1'. before removing seals.
      3. Count cells, then spin to pellet (~1200 rpm, 5').
      4. Resuspend cells at 1-5 x 106 cells/ml in serum free media.
      5. Plate 10 ul cells into wells of 384-well plate.
      6. Incubate dsRNA with cells at RT for 30'.
      7. Add 30 ul of complete media to each well.
      8. Seal the plates to prevent evaporation.
      9. Incubate 3 days and analyze.
         Length of incubation may vary depending on assay.