RNAi在细胞培养中的应用

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

I. 6-Well Plates

A. Bathing

II. 384-Well Plates

A. Bathing

B. Transfection

Ⅰ 6-Well Plates

A. 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.

Ⅱ. 384-Well Plates

A. 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 μl cells into wells of 384-well plate.

6.Incubate dsRNA with cells at RT for 30'.

7.Add 30 μl 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.

B. 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 μl cells into wells of 384-well plate.

6.Incubate dsRNA with cells at RT for 30'.

7.Add 30 μl 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.