Research article

Off-target effects of siRNA specific for GFP

Cordula Tschuch¹ , Angela Schulz¹ , Armin Pscherer¹ , Wiebke Werft² , Axel Benner² , Agnes Hotz-Wagenblatt³ , Leticia Serra Barrionuevo¹ , Peter Lichter¹ and Daniel Mertens^1,4

¹Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany

²Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany

³Division of Molecular Biophysics, German Cancer Research Center, Heidelberg, Germany

⁴Department of Internal Medicine III, University Hospital, Ulm, Germany

author email corresponding author email

BMC Molecular Biology 2008, 9:60doi:10.1186/1471-2199-9-60

Published:	24 June 2008

Abstract

Background

Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules.

Results

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.

Conclusion

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.