Properties of dsRNAs and reporter genes can influence the sensitivity of the RNAi assay. (a-c) The dynamic range of validation dsRNAs is smaller than that of the DRSC1.0 dsRNAs, which could potentially increase the rate of false negatives. The effects of dsRNA-mediated knockdown of known Wg-pathway regulators were tested by measuring their effect on the Wg reporter activity. DRSC1.0 and DRSC-v dsRNAs were compared in parallel. Knockdown of wg and arm using DRSC1.0 dsRNAs resulted in 90% and 99% reduction in Wg-reporter activity, respectively ((a), black bars). On the other hand, validation dsRNAs for wg and arm reduced reporter activity by only 58% and 90%, respectively ((a), grey bars), suggesting that the DRSC-v dsRNAs for some genes may not be as efficient in targeting their endogenous transcripts. Some of the validation dsRNAs ((b), grey and light grey bars) targeting known negative regulators did not produce robust effects on reporter activity compared to their DRSC1.0 counterparts ((b), black bars), including dlp, axn, skpA and one dsRNA in the case of slmb. Two independent validation dsRNAs targeting the same gene could influence reporter activity to different extents (compare DRSC_v1 and DRSC-v2 dsRNAs for each target gene in (c)). (d) Finally, the number of Tcf binding sites in the Wg responsive luciferase reporter gene can affect the robustness (fold change) upon induction by Wg. Reporter gene carrying 8 (white bar), 12 (grey bar) or 16 (black bar) sites were co-transfected with wg expressing cDNA. Increasing the number of Tcf binding sites increased the fold induction of the luciferase reporter upon addition of both Wg or ΔNLrp6 to induce the Wg pathway. All luciferase reporter assays were performed in 4 replicas and error bars represent the standard error between the four data points.
DasGupta et al. Genome Biology 2007 8:R203 doi:10.1186/gb-2007-8-9-r203