RESULTS Competition for RNAi components by co-transfected ShRNAs We have been interested in investigating the possibility that in the presence of multiple interfering RNAs, the saturation of RNAi components could diminish the activity of a particular sh or siRNA and generate misleading results. Most importantly, the degree of saturation could be such as to interfere with the endogeneous microRNA pathway, thus disrupting poten- tially critical regulatory functions in the cell. To test this possibility, we selected a series of shRNAs (shL, shVif, shSII, shTAT, shSI) and co-transfected each with a FLT and an anti-ﬁreﬂy-luciferase shRNA (Luc), which routi- nely generates 90% down-regulation of the target (Figure 1a). Each shRNA was also co-transfected alone with the luciferase target (constructs 2–6, Figure 1a) to monitor any non-speciﬁc reduction or increase of lucifer- ase expression. The results clearly show that there is a direct competition among non-related shRNAs (Figure 1a). The degree of competition varies with the shRNA used in the co-transfection. The down-regulation of Luc was reduced by co-transfected, non-speciﬁc shRNAs from 10 to 60%, yielding in two cases only 30% down-regulation of the luciferase target (Figure 1, a Luc+shSI and Luc+shTAT) without any targeting of RNAi components and/or presumably other cellular genes. Northern analyses of total RNA from cells transfected with these shRNA constructs did not show a correlation between the degree of competition and their intracellular expression levels (Figure 1, compare a and b). Thus, competition must be dictated by the ability of each shRNA to be processed into siRNA and incorporated into RISC. Many parameters have been analyzed in the literature that can improve shRNA designs and possibly their incorporation into RISC (28). However, none of the popular rules for the optimal shRNA design can explain the diﬀerent levels of competition exerted by each shRNA with the Luc shRNA construct (Supplementary Data). Although the relative intracellular expression of each shRNA could not by itself explain the degree of competition for each of the shRNAs, the total amount of shRNA present in the cell can render Exportin 5 rate limiting and aﬀect the overall shRNA activity. To test this possibility, we co-transfected low concentrations (either 12.5 or 50 ng) of three irrelevant shRNA expression plasmids (shL, shSII and shSI) with equal amounts of the Luc shRNA construct and its luciferase target. As expected, the higher amount of shRNAs present in the cell increased the competition while keeping the relative strength of each shRNA as a competitor unchanged (Figure 2a). The Luc shRNA showed the same ability to down-regulate the target when 12.5, 25 or 50 total ng were transfected with the luciferase target (Figure 2a, Luc-12.5, Luc-25, Luc-50). A double-stranded RNA stem-loop expressing construct (a mutated HIV TAR element that retains its structure) was also co-trans- fected with the Luc shRNA and the luciferase target to show that the competition is speciﬁc to shRNAs. The U6 Nucleic Acids Research, 2007, Vol. 35, No. 15 5157 Figure 1. Irrelevant shRNA sequences compete with a luciferase- directed shRNA and reduce its ability to down-regulate the target. (a) FLT is a U1 expressed ﬁreﬂy luciferase gene, shL is targeted against the EnvPb1 retrovirus envelope gene, shVif is targeted against the Vif gene, shSII against sequences present in the HIV-Rev, shTAT is designed against the HIV Tat gene and shSI is directed against both TAT and Rev genes. The shRNA Luc is targeted to a sequence present in the coding region of the ﬁreﬂy luciferase gene. The irrelevant shRNAs were transfected with the luciferase target to monitor non-speciﬁc eﬀects (constructs 2–6). FLT (construct 1) indicates the luciferase target transfected alone. The Bluescript vector was used to adjust the total amount of DNA transfected to be the same in each sample. Fifty nanogram of the ﬁreﬂy luciferase target and 50 ng of the shRNA constructs were used for these transfection reactions. The luciferase units were calculated by comparing each experimental sample to its own control. A Renilla luciferase plasmid was co-transfected in each sample as transfection control and used to normalize the ﬁreﬂy luciferase units. (b) Northern analysis of total RNA from 293 cells transfected with the relevant shRNAs as indicated on the top of the gel. Probes complementary to the guide sequence corresponding to each shRNA were used to detect the processed siRNA products (siRNA). A probe complementary to the endogenous U6 small nuclear RNA (U6snRNA) was used as internal control for the amount and integrity of the RNA loaded in each lane. TAR element is highly expressed in cells (not shown) yielding levels of cellular transcripts that are comparable to any of the shRNAs tested in these experiments. As shown in Figure 2a, the TAR element did not signiﬁcantly aﬀect the eﬃcacy of the Luc shRNA even when transfected at a 1:3 ratio (50 ng of Luc and 150 ng TAR). To test if the competition persists when the shRNAs are present in a linear range we performed a dose response curve using the Luc shRNA and the shSII as a competitor (Figure 2b). We used 1, 3, 5, 12.5 and 25 ng of shLuc in a luciferase down-regulation assay to select the lowest amount of shRNA that still achieved