In this protocol, we describe the feeding RNAi protocol that is routinely used in our laboratory. RNAi and isolation of deletion allele are extremely useful ways to do functional analyses of C. elegans genes. Our RNAi protocol and finer points for the success of RNAi experiments are described, but the description of the routine protocols for C. elegans culture is omitted for brevity (See WormBook at http://www.wormbook.org/ ). Our protocol is based on Timmos et al (2001), Kamath et al. (2003) and Ahringer et al. (2006) with some modifications. |
Category | Glycosyltransferases & related proteins |
Protocol Name | RNAi-mediated knocking down of gene activities in the nematode Caenorhabditis elegans by using the Ahringer’s feeding RNAi library |
Authors
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Akiyoshi, Sayaka
Graduate School of Systems Life Sciences, Kyushu University Graduate School
Nomura H., Kazuko
Graduate School of Systems Life Sciences, Kyushu University Graduate School
Nomura, Kazuya
*
Graduate School of Systems Life Sciences, Kyushu University Graduate School
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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Carbenicillin (100 mg/mL solution) |
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C. elegans RNAi library (available from Source BioScience, Nottingham, UK) |
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Instruments
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NGM (Nematode Growth Medium) agar plates |
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Incubator for worm culture (for incubation at 15–25°C) |
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A worm pick (handmade with platinum wire attached to a tip of a glass pipette) |
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A dissecting microscope (e.g. Olympus SZX-12 or SZX-16 with 1.2 ×PF ) |
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A fluorescence microscope with DIC (Differential Interference Contrast) optics (e.g. Olympus BX51 or Leica DMRXA) |
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Methods |
1. |
Checking bacterial clones for RNAi:
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2. |
Location of the bacterial clones for your desired genes in the library plates:
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3. |
Preparation for Feeding RNAi:
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1) |
Day 1: Defrost Bacteria.
Pick bacteria from the glycerol stock of the bacterial clones of your favorite genes (Replica plates of the Ahringer’s feeding RNAi library stored at −80°C are used.).
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Inoculate each of them in 1–3 mL of LB media containing 0.1 mg/mL carbenicillin.
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Culture the E. coli for 6–8 h. |
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2) |
Store the cultures at 4°C until use. |
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3) |
Seed the E. coli onto the NGM agar plates (e.g. 80 μL E. coli culture/35 mm plate, or 250 μL E. coli culture/60 mm plate), and dry the plates in a laminar flow hood under sterile condition.
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Incubate the plates at 37°C overnight. |
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4) |
Day 2: To induce the double stranded RNA synthesis with 0.2 mM IPTG, cover whole surface of each plate with the IPTG solution, and dry them in a laminar flow hood.
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Incubate the plates at 37°C for 4–6 h for dsRNA induction. |
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4. |
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Pick one L4 stage worm (this is the P0 worm) and put it onto a dsRNA induced plate prepared above.
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Let the worm eat the dsRNA induced E. coli at 25°C. See Comments. |
Comment 1
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5. |
Examination of phenotypes under a dissecting microscope
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1) |
After about 20 h, control P0 worms will grow up to gravid hermaphrodites. In some RNAi experiments, effects of RNAi are obvious in P0 worms. See comments. |
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2) |
While observing P0 phenotypes, prepare IPTG induced plates as above, and transfer the P0 worm with a worm pick onto the newly prepared plate after one day. |
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3) |
Transfer the P0 worm into freshly prepared RNAi plate twice at one day interval, and examine F1 and P0 phenotypes. |
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4) |
Score F1 and P0 phenotypes. F1 Phenotypes are usually most obvious in the second and the third plate. |
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Figure & Legends |
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Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-09-11 16:36:02 |
- Timmons, L., Court, D.L., and Fire, A. (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 263, 103–12 [PMID : 11223248]
- Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., and Ahringer, J. (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, RESEARCH0002 [PMID : 11178279]
- Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D.P., Zipperlen, P., and Ahringer J. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421, 231–237 [PMID : 12529635]
- Ahringer, J., ed. Reverse genetics (April 6, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.47.1, http://www.wormbook.org
- Qu, W., Ren, C., Li, Y., Shi, J., Zhang, J., Wang, X., Hang, X., Lu, Y., Zhao, D., and Zhang, C. (2011) Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens. BMC Genomics, 12, 170 [PMID : 21453524]
- Qadota, H., Inoue, M., Hikita, T., Köppen, M., Hardin, J.D., Amano, M., Moerman, D.G., and Kaibuchi, K. (2007) Establishment of a tissue-specific RNAi system in C. elegans. Gene, 400, 166–173 [PMID : 17681718]
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Akiyoshi, Sayaka,
Nomura H., Kazuko,
Nomura, Kazuya,
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Nomura H., Kazuko,
Nomura, Kazuya,
(2014).
RNAi-mediated knocking down of gene activities in the nematode Caenorhabditis elegans by using the Ahringer’s feeding RNAi library.
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Nomura H., Kazuko,
Nomura, Kazuya,
(2014).
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