Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive

A dual library screening strategy for the identification of targets responsible for observed phenotypes

<p><b>Copyright information:</b></p><p>Taken from "Multiplexing siRNAs to compress RNAi-based screen size in human cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):e57-e57.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1885663.</p><p>© 2007 The Author(s)</p> () The identified members of active siRNA multiplexes affecting cell viability. These target genes were identified by cross-referencing the components of the top 20 down-regulating multiplexes within library #1 with common components found in the top 20 down-regulators within library #2. () Validation of the identified targets as down-regulators of cell viability in an independent assay. All values are reported as the percent viability of cells transfected with a negative-control siRNA. As an additional reference, a multiplex found to approximate the median value in all three screens of library #1 was also used. () Evaluation of both siRNAs against the top five gene targets individually. The experiment was performed as described in (). The activity of siRNA-1 did not differ from that of the median multiplex. For each target, the activity of siRNA pairs was approximately equal to that of the more active siRNA (compare () with ())

Multiplexed siRNAs retain comparable efficacy to that exhibited by their individual counterparts

<p><b>Copyright information:</b></p><p>Taken from "Multiplexing siRNAs to compress RNAi-based screen size in human cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):e57-e57.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1885663.</p><p>© 2007 The Author(s)</p> () Two multiplexes consisting of six siRNAs corresponding to three targets (two siRNAs per target) were evaluated for their ability to reduce cognate mRNA levels as compared to the activity of respective individual siRNAs in MDA-MB-231 cells. Total siRNA concentrations were held constant for all experiments such that individual siRNAs (siRNA-1 and siRNA-2) were tested at 60 nM and multiplexed siRNAs were tested at an individual siRNA concentration of 10 nM (60 nM total). Target-specific mRNA levels were compared to those found in cells transfected with negative-control siRNA. () Same as in () except that the multiplex consisted of twelve siRNAs corresponding to six targets. In this case, the total siRNA concentration was held constant at 120 nM again using 10 nM of each individual component within the multiplex. () Same as in () except that the experiments were conducted in MCF10A cells. Similarly, multiplexes retained activity when using gene targets other than those described here or when conducting studies in additional cell lines (data not shown)

Quantification and analysis of the RNAi mediated by synthetic siRNAs targeting over 100 human genes

<p><b>Copyright information:</b></p><p>Taken from "Multiplexing siRNAs to compress RNAi-based screen size in human cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):e57-e57.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1885663.</p><p>© 2007 The Author(s)</p> () A ranking of the percentage silencing mediated by 258 siRNAs corresponding to 129 human genes. The percentage silencing was determined by comparing the mRNA levels for each gene following transfection with 50 nM target-specific siRNA to levels found in cells transfected with 50 nM negative-control siRNA. Data is shown as the mean percentage decrease in target mRNA levels observed for four independent parallel transfections (plus SD). Analysis was conducted in HCT-116 (118 siRNAs), MDA-MB-231 (138 siRNAs) and NCI/ADR-RES (two siRNAs). () The percentage decrease in mRNA and protein levels for 19 genes (two siRNAs per gene). mRNA levels were measured 48 h post-transfection, whereas total protein levels were measured 48–96 h post-transfection