Confirming specificity of RNAi in mammalian cells.

RNA interference (RNAi) is a process of sequence-specific gene silencing. Recent advances in the understanding of RNAi have provided practical tools to silence gene expression in mammalian cells, opening new possibilities for studying the functions of genes and proteins. It is important to ensure that an observed effect of RNAi is due to silencing of the intended target. Indeed, it is possible that an siRNA may silence more than one messenger RNA that is homologous in the region complementary to the siRNA. Considering that we know little about how RNAi works in mammalian cells, other artifacts may be yet to be recognized. Thus, we suggest approaches to rescue the effect of RNAi by ectopically expressing the protein of interest. These approaches involve introducing silent mutations into the complementary DNA of the protein and targeting RNAi to the untranslated regions of the gene.