Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA corresponding to nearly 90% of the 19,427 predicted genes of C. elegans (1). Feeding of this RNAi library to N2 detected phenotypes for about 10% of the corresponding genes. In an attempt to increase the number of genes with a loss-of-function phenotype, we undertook a genome-wide RNAi screen using the
rrf-3 strain.
rrf-3 mutants are hypersensitive to RNAi; they show new and more severe RNAi phenotypes compared to those obtained with N2 (2).In a preliminary experiment, we fed the clones corresponding to chromosome I and systematically compared our phenotypic data with the data previously obtained using N2 (1). These data indicated that we could increase the amount of clones with a phenotype using
rrf-3, but also that we miss phenotypes. To determine whether the differences were due to
rrf-3 or other variations, we next screened chromosome I with N2 and
rrf-3 in parallel. Using
rrf-3, we found more clones with a phenotype compared to the data with N2 from this and previous studies (1). These data confirm that
rrf-3 is more sensitive to RNAi. Furthermore, in comparing the results of the different data sets, we observed that the RNAi effect is in general variable and in particular that the frequency of false negatives can be high.The complete RNAi library was tested on
rrf-3 mutant animals. In total, we obtained results for 16,401 clones and detected RNAi phenotypes for 2077 clones (12.7%). These include 623 clones for which previously no phenotype was found, increasing the number of clones with a phenotype by 36%. In addition, there are 286 clones for which only a phenotype was found using N2 (1). The 623 extra clones we found were broadly distributed over different phenotypic classes, suggesting that
rrf-3 is in general more sensitive to RNAi. Taken together, we significantly increased the number of genes with a phenotype using
rrf-3 and confirmed many RNAi phenotypes previously found. Finally, we are using the RNAi data to systematically clone unidentified genetic mutants, and we will give an update of the results. References: 1) Kamath et al. (2003). Nature 421, 231-237. 2) Simmer et al. (2002). Curr. Biol. 12, 1317-1319.