Recent publications by several groups of researchers have suggested that small interfering RNAs (siRNA) delivered by lipid-mediated transfection induce both sequence-specific effects 1 and broad, class-specific changes in gene expression 1, 2, 3, 4. These findings challenge convictions previously held in the RNA interference (RNAi) community that assert virtual sequence specificity of siRNA knockdown, and they bring into question the value of this methodology as a research and therapeutic tool.

To test the individual contributions of siRNA and lipid delivery reagents to nonspecific gene modulation, we have compared gene-expression profiles in cells in which one of three different siRNAs targeting cyclophilin B (cyclo 1) or mitogen-activated protein kinase kinase 1, also known as MEK1 (MEK1-2 and MEK1-4), were delivered using Lipofectamine 2000 (L2K) or by electroporation. Our study demonstrates that even though the extent of target knockdown was similar for both modes of siRNA delivery (Supplementary Fig. 1 online), the number of genes modulated by each method differed markedly (Supplementary Fig. 2 online). In cells transfected with siRNA using L2K as a delivery vehicle (and using cells treated with lipid only as a reference control), 65 genes were consistently upregulated by more than 1.5-fold in all three samples (Fig. 1a and Supplementary Table 1 online), thus mimicking the broad, class-specific gene modulation described previously 1, 2, 3, 4. Subtracting the values for the lipid-only reference during the analysis did not cancel out the observed upregulation of these genes. Thus, the data could be explained by a nonspecific response induced by siRNAs, an siRNA enhancement of lipid-induced effects, or a combination of the two.