What are off-target effects and how can these be addressed in an siRNA experiment?

Highly potent siRNAs are assessed using bioinformatic approaches so that they target a unique site in a specific gene. However, as with many biological reactions, siRNA-induced silencing will generate specific and non-specific effects. In the case of siRNA, these non-specific effects can be general responses to the presence of dsRNA molecules and to transfection and experimental manipulation of the cells. Off-target effects are related to the siRNA itself and most often arise from partial complementarily of the sense or antisense strands to an unintended target. These effects are concentration dependent and therefore the best way to minimize these off-target effects is by reducing siRNA concentrations. The SMARTpool strategy takes advantage of this by ensuring lower concentrations of each individual siRNA while maintaining a higher total concentration of siRNA against the target of interest. We also suggest employing a chemical modification such as ON-TARGETplus to reduce off-target effects from both strands. Finally, bioinformatic approaches may be used to select sequences that are less likely to exhibit a high degree of off-targeting. All three strategies (pooling, modification, and bioinformatics) are combined in our pre-designed ON-TARGETplus siRNA products.