Off-target effects of siRNA specific for GFP.
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.
Tschuch
Cordula
C
Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany. c.tschuch@dkfz.de
Schulz
Angela
A
Pscherer
Armin
A
Werft
Wiebke
W
Benner
Axel
A
Hotz-Wagenblatt
Agnes
A
Barrionuevo
Leticia Serra
LS
Lichter
Peter
P
Mertens
Daniel
D
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
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins