User:
Antonio Ruiz-Vela, Spanish National Cancer Center, Madrid
Project:
Using siRNA knockdown to identify proteins involved in apoptosis
Problem:
Transfecting any siRNA may lead to off-target changes in gene expression.
Controls:
Do at least one negative control that doesn't target any endogenous gene, says Ruiz-Vela. He generally uses a scrambled version of one of his experimental siRNA sequences. Cells transfected with the scrambled siRNA should show a similar gene expression profile and phenotype as untransfected cells, but the drawback is that the scrambled siRNAs can still cause off-target effects. "There's no way to look at all the proteins in the proteome to assess whether or not you're affecting other targets," says Peter Scacheri of Case Western Reserve University in Cleveland. Running siRNA sequences through databases like BLAST can help, but because RNA interference doesn't always operate with perfect base-pair complementarity, you may not pick up all possible interacttions.
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