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Addendum

Addendum

Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila Gyeong-Hun Baeg, Rui Zhou, and Norbert Perrimon

Recently it was shown that long double-stranded RNAs (dsRNAs) can lead to "off-target effects" (OTE) in Drosophila cells (Kulkarni et al. 2006; Ma et al. 2006). We therefore generated one or two additional independent dsRNAs for each of the 121 candidate genes of the JAK/STAT signaling pathway that we initially reported (Baeg et al. 2005). Each of the newly generated dsRNAs was designed to be free of 19 base pairs (bp) or longer overlap with other genes. We retested these new dsRNAs in parallel with the original dsRNAs identified from the screen and found that 111 original dsRNAs scored, and among them, 50 could be further confirmed by one or two independent dsRNAs (Table 1). Of interest, we note that 17 of the original dsRNAs that were devoid of any 19-bp homology with other genes failed to be confirmed by additional dsRNAs. This finding suggests that other OTE rules that we have not been able to identify (such as interference with miRNA function through potential seed regions found in small interfering RNAs [siRNAs] [Lewis et al. 2003]) may also lead to false positives in large-scale screens in Drosophila cells. Alternatively, it is possible that knockdown efficiency varies among different long dsRNAs. In addition, nine of the dsRNAs in the initial 121 positives that, based on our in silico analysis, were predicted to have off-target sequences targeting 15 or more other genes could be confirmed with a second or third dsRNA. Taken together, our data strongly support the recommendation made by Echeverri et al. (2006) that testing of two or more independent dsRNAs should be performed, and will help minimize the risk of reporting false positives in RNA interference (RNAi)-based assays. In conclusion, cell-based assays and RNAi, when well controlled, constitute a valid approach for identification of genes potentially involved in a given biological process, but more detailed biochemical and genetic analyses will be necessary to validate these candidate genes.

	References

Top References

Echeverri, C.J., Beachy, P.A., Baum, B., Boutros, M., Buchholz, F., Chanda, S.K., Downward, J., Ellenberg, J., Fraser, A.G., Hacohen, N., et al. 2006. Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3: 777–779.[CrossRef][Medline]

Kulkarni, M.M., Booker, M., Silver, S.J., Friedman, A., Hong, P., Perrimon, N., and Mathey-Prevot, B. 2006. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3: 833–838.[Medline]

Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P., and Burge, C.B. 2003. Prediction of mammalian microRNA targets. Cell 115: 787–798.[CrossRef][Medline]

Ma, Y., Creanga, A., Lum, L., and Beachy, P.A. 2006. Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443: 359–363.[CrossRef][Medline]

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This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Search for Related Content Social Bookmarking                   What's this?