Titrate siRNAs. Using the lowest possible concentration of siRNA may help avoid unwanted side effects, says Taylor. "Off-target effects can be dose-dependent." Before you start your study, run experiments with different siRNA concentrations to determine the level of knockdown you need to see a change in phenotype, Behlke says.
Optimize for each cell type. "The efficiency of siRNAs to enter the cell and target mRNA seems to differ between different types of cells," says Jaap van Buul of the University of Amsterdam. Even if you've purchased siRNAs whose knockdown levels have been validated by the company that sells them, "it's very important to study what level of knockdown is actually achieved in your hands with your cell line," Behlke says.
Watch out for innate immune responses. siRNAs can trigger inflammatory responses, especially through the Toll-like receptor pathway. "Certain siRNAs I've used have stimulated quite a lot of pro-inflammatory cytokine production," ...
