Off-Target Analysis of Control siRNA Molecules Reveals Important Differences in the Cytokine Profile and Inflammation Response of Human Fibroblasts

The use of RNA interference for the manipulation of gene expression has seen great applications from basic science to clinical investigations. However, limited selectivity and the induction of off-target effects by double stranded RNA molecules have been analyzed and discussed since the discovery of this gene expression regulation mechanism. In this study, the specificity of 13 commercially available control siRNA molecules is addressed by the analysis of gene expression profiles in 2 human cell lines HT1080 and HaCaT and in the mouse cell line 3T3-L1. The off-target signatures of the transfected siRNA molecules differ greatly between the cell lines and only a small overlap was seen for the 2 human cell lines. In particular, the HT1080 cell line showed the highest number of detected gene expression differences. In these cells, several different control siRNA molecules activated a common profile of 79 deregulated genes including a reduced interleukin-1 beta (IL-1 beta) and IL-24 expression. Functional analysis of MMP1 secretion and tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release revealed a reduction of NF kappa B signaling caused by at least 2 out of the 13 tested control siRNA molecules. Our findings strongly argue for a careful analysis of the control siRNA molecules for any given RNAi experiment.