Drosophila RNAi Screening Center

DRSC >> Protocols >> RNAi

RNAi Protocols

  1. 6-Well Plates
    1. Bathing
  2. 384-Well Plates
    1. Bathing
    2. Transfection
    3. Transfection set up for 384- and 96-well plates

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.

  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.

    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.



  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. Put the plate lid on the plate, and place the plate in a plastic container lined with damp paper towels. Put the top on the container without closing it all the way. This will better maintain local humidity around the plate during incubation.
      9. Incubate 3 days and analyze.
        Length of incubation may vary depending on assay.


    2. Transfection (Using Qiagen’s Effectene™ as the transfection reagent )
      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. Count cells and dilute them in fresh medium so that the desired number of cells is in 30-40 µL of medium that can be dispensed in each well.
      3. Centrifuge the plates at 1200 rpm. Remove the sealing foil. Remove the water and then add 5µl of ~0.016µg/µl control dsRNA to the dedicated water wells in the 384-well plate.
      4. Prepare EC + DNA Master Mix. The master mix for a 384-well plate consists of all DNA constructs that need to be introduced into cells (which may include the experimental reporter, a ligand expression vector, and the control reporter) and EC buffer, a component of the Effectene kit. Dilute the DNA into 8.75 µL EC buffer. It is best if the volume of all DNA solutions represent less than 3% of the final volume of the master mix. If this is not possible, we recommend to precipitate the DNA material and to resuspend it directly in EC buffer. It is important to keep the nucleic concentration equal in all wells and use no more than 175 ng of total nucleic acid (DNA + dsRNA) per 384 well. Mix EC + DNA well by inversion or pipetting.
      5. Add Enhancer to the EC+DNA master mix such that there is 1.0 µL of Enhancer per well. Mix well by inversion. Incubate at RT for 2-3'.
      6. Prepare the transfection solution by adding Effectene to the Enhancer + EC+DNA master mix such that there is 0.3 µL of Effectene per well. Mix by pipetting up and down 5-10 times or vortexing on high for 10". Immediately begin dispensing the transfection solution into the assay plates.
      7. Let the dsRNA and transfection solution incubate in the well for 4-8 minutes.
      8. After 4-8 minutes, dispense the desired number of cells in the predetermined volume of medium into the 384 well plate. It is critical to get the transfection solution and the cells into the wells within 20 minutes of making the master transfection solution, or the transfection efficiency begins to fall rapidly.
      9. Incubate the plates in a humidified chamber in a TC incubator for 3-5 days, then conduct assay.
        Length of incubation may vary depending on assay.

    3. Transfection set up for 384- and 96-well plates
      Plate
      Diameter
      (mm)
      Area
      (mm2)
      Fold Difference in
      Area from 96 well
      Fold Difference in
      Area from 6 well
      DNA per well
      (µg)
      Cells per well
      (x 104)
      384
      3.5 Square
      12.25
      1/2.6
      1/80
      0.050-0.175
      1-3 x 104
      96
      6.4
      32
      1
      1/30
      0.20-0.30
      3-8 x 104


      Plate
      Diameter
      (mm)
      Area
      (mm2)
      EC buffer
      (µl)
      Amount enhancer
      (µl)
      Amount Effectene
      (µl)
      Medium (with cells)
      added to well (µl)
      384
      3.5 Square
      12.25
      8.75
      1.00
      0.30
      30-50
      96
      6.4
      32
      21.5
      2.50
      0.75
      100-150