title: Large-scale gene knockdown in C. elegans using dsRNA feeding libraries to generate robust loss-of-function phenotypes.
abstract: RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.
authors.affiliation: Molecular and Cellular Biology Program, University of Massachusetts, Amherst.
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: Maher
authors.forename: Mary
authors.initials: M
authors.lastname: Catanese
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Chase
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D055785:Gene Knockdown Techniques / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000008:administration & dosage* / Q000235:genetics
chemical_list: D012330:RNA, Double-Stranded