title: Off-target effects of siRNA specific for GFP.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany. c.tschuch@dkfz.de
authors.forename: Cordula
authors.initials: C
authors.lastname: Tschuch
authors.forename: Angela
authors.initials: A
authors.lastname: Schulz
authors.forename: Armin
authors.initials: A
authors.lastname: Pscherer
authors.forename: Wiebke
authors.initials: W
authors.lastname: Werft
authors.forename: Axel
authors.initials: A
authors.lastname: Benner
authors.forename: Agnes
authors.initials: A
authors.lastname: Hotz-Wagenblatt
authors.forename: Leticia Serra
authors.initials: LS
authors.lastname: Barrionuevo
authors.forename: Peter
authors.initials: P
authors.lastname: Lichter
authors.forename: Daniel
authors.initials: D
authors.lastname: Mertens
mesh_terms: D002460:Cell Line; D015536:Down-Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry*; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins