Off-targeting remains a central challenge in the application of RNAi knockdown experiments utilizing si- or shRNAs. Especially if it comes to screening in medium- to large scales off-targeting increases cost and efforts by reducing validation rates. Off-targeting means that the used RNAi mediator silences other transcripts in addition to the intended one in a sequence dependent manner. Consequently, observed phenotypes can be due to the knockdown of the intended target, the unintended silenced transcripts or to a combination of both.
Some improvements to increase the specificity of siRNAs have been achieved, e.g. by avoiding the 3’-untranslated regions or seed-region homologies. But a design algorithm that can faithfully predict and prevent off-target effects is presently not available.
In contrast, esiRNA are inherently target-specific due to the complex mixture of many different siRNAs. Pooling of many siRNAs that all share their on-target gene is known to increase specificity because all siRNAs in the pool differ by their sequence-dependent off-target signatures. As a consequence, all individual siRNAs add to the overall silencing of the target, while off-target effects are diluted out (figure 2a). A quantitative correlation between pool complexity and specificity can be seen as well. Measuring the number of off-target transcripts by microarray after transfection of increasingly complex pools of siRNAs conclusively shows this quantitative relationship (figure 2b and 2c).
In order to evaluate the performance of esiRNAs, we compared the silencing specificity of DEQOR-optimized esiRNAs with chemically synthesized siRNAs using microarray expression analysis. EsiRNAs showed more than 10 fold higher target specificity in comparison to individual siRNAs (figure 3a, 3b), making esiRNAs the silencing trigger of choice for transcript silencing with minimal off-target effects.
All esiRNAs are optimized by the design algorithm DEQOR, which allows enrichment with highly potent siRNAs in an esiRNA-pool. EsiRNAs are therefore highly efficient in target-gene silencing as proven by QPCR and Western Blotting (figure 4a and 4b). Thereby pooling omits the elaborate and time-consuming search for efficient silencing triggers. Make esiRNAs your RNAi trigger of choice for efficient and specific RNAi experiments.
