RNA Interference (RNAi) by Bacterial Feeding   

Abstract

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.