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| The introduction of double-stranded RNA into C. elegans animals induces an effective and specific disruption of gene expression. The effect wears off however, because the double-stranded RNA is not inherited. The contributor of this protocol found that greater than 90% of the worms displayed a RNAi phenotype when testing an embryonic lethal phenotype. |
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A. Making Double Stranded RNA (dsRNA) for the Gene of Interest (see Hint #2)
1. Clone the cDNA or exons corresponding to the gene of interest into pBluescript or other appropriate vector that contains bacterial polymerase promoter sequences flanking the cloning site (i.e. T3, SP6, and/or T7).
2. Perform two separate restriction digestion reactions with the appropriate Restriction Enzymes (one adjacent to the 3' end of the cloned gene, and one adjacent to the 5' end) to linearize the vector (see Hint #3).
3. Once the reactions are completed, add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (50:49:1). Mix vigorously for a few seconds by vortexing.
4. Centrifuge at maximum speed in a microcentrifuge for 5 min to separate the aqueous and organic phases. Remove the aqueous phases to new tubes.
5. Add 0.1 volume of 3 M Sodium Acetate and 2.5 volumes of 100% Ethanol to precipitate the DNA. Mix by inverting the tubes a few times.
6. Incubate the DNA at -70°C by keeping the tubes on Dry Ice until the samples are solid (alternatively, precipitate at -20°C overnight).
7. Pellet the DNA by centrifuging at maximum speed in a microcentrifuge for 10 min.
8. Discard the supernatant, and wash the pellets with 70% Ethanol by adding 500 μl of 70% Ethanol to each tube and mixing by inversion.
9. Centrifuge at maximum speed in a microcentrifuge for 5 min. Remove the supernatant and air dry the DNA pellets.
10. Resuspend the DNA in DEPC-ddH2O to a concentration of 1 mg/ml.
11. Perform the appropriate in vitro transcription reactions to produce sense and antisense transcripts of the gene of interest (see Hint #4).
12. Confirm that the transcription reactions were successful by loading a small sample (i.e. 1 μl of a 20 μl reaction) on an Agarose Gel and staining the gel with Ethidium Bromide to visualize the transcripts (see protocol on Agarose Gel Electrophoresis).
13. Add 1 μl of RNase-free DNase to each of the transcription reactions.
14. Incubate for 15 min at 37°C to digest the plasmid DNA.
15. Incubate the sense and antisense transcripts at 65°C for 15 min in a heat block to inactivate the DNase enzyme.
16. In a new tube, mix equal molar amounts of sense and antisense transcripts and incubate again at 65°C for 15 min.
17. Remove the block from the heat source, and allow the block to cool slowly to room temperature to allow the complementary sense and antisense RNA to anneal.
18. Centrifuge the annealed dsRNA at maximum speed in a microcentrifuge for 5 min at 4°C.
19. Remove the supernatant to a new tube and store this supernatant at -20°C (see Hint #5).
B. Small Scale (approximately 5 to 50 worms) Preparation
1. Dilute the dsRNA in 100 μl of M9 Buffer to a final concentration of 100 ng/μl (see Hint #6).
2. Pick individual L4 or young adult worms from the plate and transfer each worm directly to the microcentrifuge tube containing the dsRNA. Collect up to 50 animals (see Hint #7).
3. Poke a small hole in the cap of the microcentrifuge tube using an 18-gauge needle. Cap the tube.
4. Incubate the tube at 20°C overnight.
5. After the incubation, transfer the worms from the microcentrifuge tube to fresh worm plates with a pipette tip and pipetteman (see Hint #8).
6. Incubate the plate of soaked worms for 24 hr at 20°C to allow the worms to recover.
7. Examine the worms for the desired RNAi phenotype.
C. Large Scale (more than 50 worms) Preparation
1. Grow large plates of worms to obtain many gravid adults.
2. Perform a standard Embryo Preparation (see Protocol on C. elegans Embryo Preparation).
3. After allowing embryos to develop into L4-young adult stage worms, wash worms off plates, using 5 ml of M9 buffer per plate.
4. Collect each 5 ml of worms in M9 buffer in individual 15 ml conical centrifuge tubes.
5. Pellet the worms at 700 X g for 1 min.
6. Resuspend each pellet in 1 ml of M9 buffer and transfer each 1 ml to a well of a 12-well tissue culture plate.
7. Add dsRNA to a final concentration of 50 to 500 ng/μl per well (see Hint #6).
8. Place the culture plate on a shaking platform and incubate at 20°C overnight with gentle shaking (see Hint #9).
9. After incubation, transfer the worms for each well to a large worm plate and allow them to recover overnight at 20°C.
10. Examine worms for phenotypes or continue with analysis via conventional molecular and cell methods.
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| DEPC Treated ddH2O |
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| DEPC Treated ddH2O |
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| DEPC Treated ddH2O |
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| DEPC Treated ddH2O |
| Shake vigorously to mix, and let stand overnight
Autoclave to inactivate the DEPC (CAUTION! see Hint #1)
Add Diethylpyrocarbonate (DEPC)to 0.1% (v/v) in ddH2O
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| 70% (v/v) Ethanol |
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| 3 M Sodium Acetate |
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| Phenol:Chloroform:Isoamyl Alcohol |
| 50:49:1Phenol:Chloroform:Isoamyl Alcohol
CAUTION! see Hint #1
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| M9 Buffer |
| 22 mM Potassium Phosphate Monobasic (KH2PO4)
19 mM NH4Cl
48 mM Sodium Phosphate Dibasic (Na2HPO4)
9 mM NaCl
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| DEPC Treated ddH2O |
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| DEPC Treated ddH2O |
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| DEPC Treated ddH2O |
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Chloroform
Restriction Enzyme
Phenol
DNase, RNase-free
Potassium Phosphate, Monobasic
In Vitro Transcription Kit
Sodium Phosphate, Dibasic
Ethanol
DEPC
Sodium Acetate
Urea
Agarose
Sodium Chloride
Acrylamide
Ammonium Chloride
Isoamyl Alcohol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC (diethyl pyrocarbonate) to inactive RNases and use solutions prepared with RNase-free water and equipment. For further information on working with RNA see Section on working with RNA under Useful Stuff in the main menu.
3. Linearization will allow for transcription of the gene of interest such that transcripts will be produced in both the "sense" and "antisense" orientations.
4. Kits are also available for in vitro transcription; the contributor of this protocol suggests using a kit by Ambion.
5. To produce pure dsRNA, follow Step #19 with a Phenol/Chloroform extraction and Ethanol precipitation.
6. A titration may be helpful to determine the optimal concentration of the particular gene of interest's dsRNA that results in maximum inhibition. Start with a minimum of 50 ng/ml.
7. This method does not seem to work with egl mutants.
8. The worms may look a little sick at this point, but they generally recover.
9. You do not have to agitate very much, just enough to keep the worms oxygenated.
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