Double Stranded RNA Interference (dsRNAi) in Drosophila Schneider S2 Tissue Culture Cells
Nancy Leff of the Laboratory of Jack Dixon at the University of Michigan.
Overview
This protocol describes the preparation of double-stranded RNA (dsRNA) and inhibition of gene
expression with exposure of Drosophila tissue culture cells to the prepared dsRNA (dsRNAi). The
procedure employs a commercial kit (Ambion) to produce quantities of RNA sufficient for the
treatment of 10 million cells. In this procedure, oligonucleotides containing the T7 RNA
polymerase promoter are used for PCR amplification of a fragment of the gene of interest. The
resulting product contains a T7 promoter at each end so that it may be transcribed with T7 RNA
polymerase to produce RNA transcripts corresponding to both strands of the template. Subsequent
hybridization of the complementary transcripts produces dsRNA. Treatment of cells with the
dsRNA typically results in 95% to 99% inhibition of expression of the target gene.
Procedure
A. Preparation of dsRNA
1. Design the two oligonucleotides such that the 5' portion encodes a T7 RNA polymerase
promoter and the 3' portion is complementary to one strand of the template cDNA. Choose the
oligonucleotides such that the PCR product will be between 300 and 1000 basepairs in length
(see Hint #1).
2. Amplify a PCR product from a cDNA clone containing the gene of interest using the two
oligonucleotides as described in Protocol ID#213.
3. Purify the PCR product as described in Protocol ID#1040. Alternatively, use one of the
many commercially available products for PCR purification (see Hint #2) or extract the DNA
with Phenol/Chloroform followed by Ethanol precipitation.
4. Resuspend the PCR product at a concentration of 0.125 mg/ml or higher in DEPC-treated
ddH2O (see the BioTools section of the website). To determine the concentration of the PCR
product, electrophorese a sample of the product on a 1% Agarose gel as described in Protocol
ID#1265.
The following steps are designed for use with the Ambion MegaScript™ RNA synthesis kit.
Other T7 RNA Polymerase reaction kits can also be used, following the manufacturer's
instructions.
5. Thaw the reaction components, except Enzyme mix, at room temperature (see Hint #3).
6. Add the reagents from the kit in the following order into a 1.5 ml microcentrifuge tube at
room temperature:
X μl of DEPC-ddH2O to a final volume of 20 μl
2 μl of 10X Reaction Buffer
2 μl each of rATP, rCTP, rGTP, rUTP mixes
X μl of linearized DNA template (1 μg total)
2 μl of Enzyme mix
Flick the tube a few times to mix the tube gently then incubate the reaction at 37°C for 2 to
6 hr.
7. Add 80 μl of ddH2O, 10 μl of 3 M Sodium Acetate, and 250 μl of Ethanol to the reaction to
precipitate the nucleic acid.
8. Mix the reaction and incubate the tube on dry ice for 15 min.
9. Centrifuge the tube at maximum speed in a microcentrifuge for 15 min.
10. Discard the supernatant.
11. Air-dry the pellet of RNA for 15 min.
12. Resuspend the pellet in 40 μl or less of DEPC-ddH2O (see Hint #4).
13. Heat the resuspended RNA at 65°C for 30 min. Place tube in a beaker of water at 65°C
and place the beaker on the bench to cool the water slowly to room temperature so that the
RNA strands are able to anneal.
14. Run 1 to 2 μg of the dsRNA on a 1% Agarose gel to check the integrity and size of the
dsRNA (see Hint #5).
15. Store the dsRNA at -80°C (see Hint #6).
B. Incubation of Cells with dsRNA
1. Plate Drosophila S2 cells into 6-well, 35 mm dishes in Serum-Free Expression Medium at a
concentration of 106 cells/ml/well.
2. Immediately add 15 μg of dsRNA.
3. Swirl the plates for 10 sec to mix RNA and cells.
4. Incubate the cell suspension at room temperature for 30 to 60 min.
5. Add 2 ml of Complete Medium and return the plates to the incubator at room temperature
(see Hint #7).
6. Incubate the cells for 24 to 48 hr.
7. Use a rubber policeman to dislodge and harvest the cells by scraping and transferring them
directly from the dish into 1.5 ml microcentrifuge tubes.
8. Pellet the cells by centrifugation at 200 X g for 5 min. Discard the supernatant.
9. Resuspend the cells in 1 ml of RIPA Buffer and triturate for 30 sec to lyse the cells.
10. Centrifuge the lysates at maximum speed in a microcentrifuge for 20 min at 4°C.
11. Transfer the supernatant to a fresh microcentrifuge tube.
12. Check the level of the protein of interest from the extract prepared in Step #11 by
SDS-PAGE/Immunoblotting or by Immunoprecipitation (see Protocol ID#455, Protocol
ID#70, Protocol ID#315; see Hint #8).
Solutions
RIPA Buffer
1% (v/v) Igepal CA630 (NP-40; Sigma)
Store at 4°C
Filter to sterilize
0.5% (w/v) Deoxycholate
150mM NaCl
10 mM Sodium Fluoride (CAUTION! See Hint #9)
50 mM Tris-Cl, pH 8.0
0.4 mM EDTA
0.2 mM Sodium Vanadate
10% (v/v) Glycerol
0.1% (w/v) SDS
DEPC-ddH2O
0.1%(v/v) Diethylpyrocarbonate (CAUTION! See Hint #9)
See the BioTools section of this site for preparation
3 M Sodium Acetate Complete Medium
100 Units/ml Penicillin
10% (v/v) Fetal Bovine Serum
Filter to Sterilize
100 μg/ml Streptomycin
Store at 4°C
Schneider's Drosophila medium (Gibco/BRL)
Serum-Free Expression Medium
2 mM Glutamine
Store at 4°C
DES Serum-free Expression medium (Invitrogen)
Filter to sterilize
BioReagents and Chemicals
Sodium Acetate
Fetal Bovine Serum
Sodium Vanadate
Glutamine
Igepal CA630
SDS
Deoxycholate
Diethylpyrocarbonate
Glycerol
Sodium Fluoride
Tris-Cl
Streptomycin
Penicillin
Sodium Chloride
EDTA
Protocol Hints
1. The T7 RNA polymerase promoter sequence is as follows:
5'-TTA ATA CGA CTC ACT ATA GGG AGA-3'
2. The contributor of this protocol has used the High Pure PCR Product Purification kit from
Boehringer with success.
3. Do not place the components on ice as precipitates may form if the reagents are chilled.
4. The dsRNA should be at a final concentration of 3 μg/μl. Take a sample of the RNA and
measure the optical density of the sample at 260 nanometer wavelength (OD260). Calculate the
concentration based on an OD260 of 1 equal to 45 μg/ml RNA.
5. A typical reaction yields 150 μg to 200 μg of dsRNA.
6. dsRNA appears to be stable for a minimum of 4 months at -20°C, with no loss of activity.
Storage at -80°C will preserve the dsRNA for even longer periods of time.
7. Schneider S2 cells are grown in an incubator at room temperature without carbon dioxide. S2
cells grow in suspension in spinner flasks, or attached as monolayers on plates and dishes.
8. The contributor of this protocol provides answers to frequently asked questions (FAQ) at the
following website: Dixon Lab Protocol Page.
9. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper
handling instructions.