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RNA Interference (RNAi) by Bacterial Feeding   

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Abstract

There are 3 ways to perform RNAi in worms: microinjection, soaking and feeding. In the feeding protocol, RNAi is induced by cultivating worms on bacteria expressing gene-specific dsRNA. dsRNA is expressed in E. coli and ingested by worms. This protocol describes the feeding protocol to induce RNAi.

Materials and Reagents

  1. C. elegans RNAi clone/libraries (Open Biosystems or Source BioScience LifeSciences)
  2. Ampicillin (IBI Scientific)
  3. Tetracycline
  4. IPTG (Gold Biotechnology)
  5. LB agar medium: Agar (BD Biosciences), tryptone (BD Biosciences), yeast extract (BD Biosciences), NaCl (Research Organics)
  6. RNAi plate (NGM/IPTG/Ampicillin) (see Recipes)

Equipment

  1. Aluminum foil
  2. Petri dish (35 x 10 mm)

Procedure

  1. Streak dsRNA-expressing E coli onto LB agar plate containing ampicillin (50 μg ml-1) and tetracycline selection (12.5 μg ml-1) and incubate at 37 °C overnight.
    Note: There are two RNAi feeding libraries. One was constructed by the Vidal and Heuvel lab and can be ordered from Open Biosystems; the other was constructed by Julie Ahringer's lab and is available at Source BioScience LifeSciences.
  2. Inoculate bacteria in 3 ml LB liquid medium containing ampicillin (100 μg ml-1) only and incubate at 37 °C overnight.
  3. Spin down all 3 ml culture and pour off supernatant to 150 μl (concentrate culture by 20x). Resuspend pellet.
  4. Transfer 50 μl of cell resuspend to center of RNAi plate (NGM/IPTG/Ampicillin). Let dry (wrapped in aluminum foil) and induce overnight at room temperature (RNAi-seeded plates can be stored at RT for 2-3 days before use).
  5. Place 10-15 egg-laying worms on each plate. Incubate 2 - 6 h at 20 or 25 °C. Suck off parents and incubate at desired temperature until desired stage for further experiments.
    Note: Instead of egg lay, synchronize worms by bleach and transfer starved L1 larva to RNAi plates.

Recipes

  1. RNAi plate (NGM/IPTG/Ampicillin)
    Use the same recipe for making NGM agar medium but instead of adding streptomycin, add IPTG to final a concentration of 1 mM and ampicillin with a concentration of 100 μg ml-1. Typically pour onto small petri dish (35 x 10 mm).

References

  1. Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395(6705): 854.
  2. Rual, J. F., Ceron, J., Koreth, J., Hao, T., Nicot, A. S., Hirozane-Kishikawa, T., Vandenhaute, J., Orkin, S. H., Hill, D. E., van den Heuvel, S. and Vidal, M. (2004). Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14(10B): 2162-2168.
  3. Kamath, R. S. and Ahringer, J. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30(4): 313-321.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). RNA Interference (RNAi) by Bacterial Feeding. Bio-101: e59. DOI: 10.21769/BioProtoc.59.
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