Silencer Select siRNA Design Algorithm Significantly Improves Effective siRNA Prediction Accuracy. The Silencer Select siRNA design algorithm was used to design 155 siRNAs to 40 different targets. These siRNAs were tested side by side with siRNAs designed using the previous algorithm at 5 nM in HeLa cells. mRNA knockdown was measured 48 h post-transfection via qRT-PC R using TaqMan® Gene Expression Assays. Results are expressed as percent of mRNA remaining compared to Silencer Negative Control #1 siRNA treated cells. The inset shows the percentage of siRNAs that elicited ≥70% and ≥80% mRNA knockdown.

Silencer Select siRNAs Provide Up to 100X Higher Potency Compared to Other siRNAs. Silencer Select siRNAs to 10 different targets and siRNAs from two other suppliers to the same 10 different targets were individually transfected into HeLa cells in triplicate at the indicated siRNA concentration. mRNA knockdown levels were tested 48 h later as described in Figure 1. Average percent mRNA remaining is shown for each set of siRNAs.

Silencer Select siRNAs Elicit Expected Phenotype at a Higher Rate than Other siRNAs. siRNAs to seven gene targets with well understood RNAi induced phenotypes were individually transfected at 3 nM and phenotypes measured 48 hours later. Each bar represents the percent of siRNAs that gave the expected, silenced phenotype. siRNAs to BUB1B, AURKB, WEE 1, and PL K1 were assessed using a multi-parametric cell growth / apoptosis assay in U2OS human osteosarcoma cells. siRNAs to HMGCR, LDLR, and FDFT 1 were assessed using an LDL uptake assay in HUH7 human hepatoma cells.

Five-step Bioinformatic Filtering Process that Eliminates siRNAs
Predicted to Elicit Off-target Effects

Luciferase reporter gene constructs with siRNA targets cloned in either the sense (guide strand target) or antisense (passenger strand target) orientation were co-transfected with the corresponding siRNA and a ß-galactosidase encoding control vector. Luciferase and ß-galactosidase assays were performed 72 hours later, and knockdown for each strand was calculated relative to negative control siRNA transfected cells. (A) The ratio of guide to passenger strand knockdown is shown for 46 siRNAs with and without the Silencer Select modifications. (B) The average ratio of guide to passenger strand knockdown is plotted versus luciferase knockdown for 6 Silencer Select and 36 competitor siRNAs.

Silencer Select siRNA Modifications Reduce the Number of Off-target, Differentially Expressed Genes. Three negative control siRNAs with and without the Silencer Select modifications were individually transfected in quadruplicate into HeLa cells at 30 nM. RNA was extracted and analyzed on an Affymetrix Human Genome U133 Plus 2.0 Array in triplicate. The y-axis indicates the average number of differentially expressed genes — those showing ≥2-fold change in expression versus mock transfected samples

Silencer Select siRNA Modifications Reduce Off-target Effects and Yield More Reliable Phenotypic Data. 53 different siRNAs, including older designs previously noted to elicit offtarget phenotypes, were transfected into U2OS cells at 30 nM in both unmodified and Silencer Select modified formats. Mitosis and apoptosis were measured 48 hours later. Data is expressed relative to negative control siRNA transfected cells. Black = similar mitosis/apoptosis levels as control. Green = down-regulation. Red = up-regulation. Note that the expected mitotosis and apoptosis phenotypes for PLK and WEE1 siRNAs are preserved with the modifications. In contrast the off-target apoptotic phenotypes elicited by 10 unmodified siRNAs were completely eliminated with addition of the Silencer Select modifications.