What are the key parameters contributing to unwanted off-target effects in an RNAi experiment?

“Off-target effects” refers to any molecular and/or cellular change that occurs during the experiment that is not attributable to the sequence-specific RNAi event under study. There are two primary causes of off-target effects. The most common cause is utilizing an inferior siRNA/shRNA design algorithm. It is absolutely critical that a rigorous design algorithm be utilized, in order to assure gene specificity and maximal efficacy in your RNAi experiments. The SureSilencing shRNA plasmids have been engineered according to a proprietary, experimentally verified algorithm. The SureSilencing shRNA plasmids have been shown to out-perform the current TRC (The RNAi Consortium) collections, which serve as the basis for most of the commercially available shRNA product lines. For a detailed description of this study, please visit the following web address:



In addition to poorly-designed siRNA/shRNA molecules, the transfection conditions utilized in an experiment commonly contribute to off-target effects. This is one of the reasons that it is critical to include reliable, validated negative controls in your RNAi experiment. We recommend simultaneously monitoring transfection efficiency, gene knock down efficacy, and off-target effects (cell viability and proliferation, and global gene expression patterns) when carrying out transfection optimization studies (see the next FAQ on optimizing transfection conditions). By carrying out these rigorous process development studies, you can find the ideal conditions where maximal efficacy and minimal off-target effects are observed.


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