RNA?interference?protocol

We use the Promega Ribomax Large Scale RNA Production System T7 (Cat. No.: P1300) to produce dsRNAs and the RNAi Exp.html" target=_blank> Jack Dixon protocol ( RNAi _Dixon.html" target=_blank>downloaded version,15.1.2003 ) to prepare dsRNAs.

I. Preparation of Template DNA

1.Design two oligos for your gene of interest.

Each should incorporate a 5' T7 RNA polymerase binding site, resulting in a PCR product of approximately 700 bp.

2.T7 RNA polymerase binding site: TTA ATA CGA CTC ACT ATA GGG AGG

3.Purify the PCR DNA template

It should be free of RNases and inhibitors such as high salt, detergents or EDTA.

4.Quantify the PCR Product on an agarose gel.

The dsRNA reaction detailed below requires 5-10 µg total DNA in 40 µl.

II. Preparation of the dsRNA

We use the Promega Ribomax Large Scale RNA Production System T7. We modify the supplied protocol as follows:

1.Prepare the templates as described above.

2.Add the following reagents from the kit in a 1.5 ml Eppendorf tube at RT. For a 100µl reaction:

20 µl Buffer 5x

30 µl rNTPs (25 µM)

DNA 5 µg

10 µl T7 enzyme mix

nuclease free water to a final volume of 100 µl

3.Mix tube gently, spin contents down and incubate at 37?C for 2 to 6 hours or overnight

4.Add 2 µl of RNase free Dnase. Incubate for 1 h at 37?C . Add 200 µl of nuclease free water and extract the RNA with Phenol:Chloroform

5.Ethanol precipitate your reactions

6.Resuspend the pellet in 100 µl - 200 µl water.

7.Heat your reactions tubes at 70?C for 30 min.

8.Place tubes in a beaker of 80?C water on the bench and slowly cool to room temperature to anneal the RNA.

9.Adjust the final concentration of your dsRNA to 3 µg/µl, using the following calculation: OD260nm 1 = 45 µg/ml

10.Run 1-2 µg of your dsRNA on a 1% Agarose gel to check the integrity and size of the dsRNA. A 100 µl reaction typically yields up to 1 mg of dsRNA.

III. RNAi in Drosophila Schneider cells.

1.Wash your Drosophila cells with serum free medium and plate them into a big flask with 2x106 cells/ml.

2.Add 15 µg dsRNA to per 106 cells (notice: check for knock down efficiency and use appropriate amounts of dsRNA)

3.Swirl flasks well to mix RNA and cells.

4.Incubate for 1-3 hours in the incubator.

5.Add the same volume of Drosophila Schneider Medium supplemented with double amounts of Fetal Calf Serum, Penicillin/Streptomycin and Glutamine to bring the medium back to normal growth conditions.

6.Harvest 3-4 days later, depending on your knock down efficiency.