Off-target effects of siRNA specific for GFP

We tested the most widely used control siRNA directed against GFP for off-target effects and found that it deregulates in addition to GFP a set of endogenous target genes. The off-target effects were dependent on the amount of GFP siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for GFP is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes.We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against GFP. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used GFP siRNA as a reference resource for future siRNA experiments.RNA interference (RNAi) is a powerful method to specifically suppress gene expression and is therefore widely used for experimental as well as therapeutic purposes. Since the initial characterization of RNAi in the nematode C. elegans [1], the field of RNAi has expanded remarkably. The essentials of RNAi can be summarized as specific degradation of target mRNA mediated by small, double stranded RNAs [2,3]. Meanwhile, different mechanisms of RNAi were discovered in mammalian cells comprising post-transcriptional gene silencing by siRNAs, transcriptional silencing by siRNAs in the nucleus and the microRNA pathway [4,5]. It has been shown that exogenously introduced siRNA duplexes and endogenously processed miRNA duplexes are taken up by the RNA-ind