Tips for perfect siRNA controls

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," Taylor says. Examining expression levels of known immune genes - such as downstream targets of interferon-α - can reveal signs of the problem. Also, cationic lipids, often used to help transfect siRNAs into cells, may cause an immune response, she adds, so if you're using one, "you should be sure to also include a mock transfection control to monitor the cellular effects of the transfection process." You can also buy siRNAs that are chemically modified to evade immune response, Behlke says, which is especially important in vivo. The only reason not to use these modified siRNAs routinely is that they're more expensive. "In most cell lines, it's not necessary."

Do positive controls every time. While some researchers perform only a positive control in the beginning, "You need to know how much you're knocking down your gene every time," Behlke says. It's possible that transfection efficiency could change, for unknown reasons. If the positive control isn't knocked down to the expected level, Behlke throws out the data for that trial.