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SiRNA profiling of nu/nu after GFP targeted knockdown to examine off target effects of GFP siRNA

ABSTRACT: siRNA mediated gene knockdown has been shown to be extremely sequence specific. However, off-target gene regulation due to partial sequence homology has also been reported. Furthermore, sequence unrelated effects on gene regulation can occur when siRNA is applied in vivo. In this experiment we investigated off target gene regulation due to treatment of mice with siRNA and LNA modified siRNA targeting GFP. Off target gene regulation was observed and was markedly reduced upon introduction of LNA modifiecation in siRNA. Experiment Overall Design: NMRI nu/nu mice were treated with saline (control), siRNA or siLNA for three weeks. Then the mice were sacrificed and tumor total RNA was isolated. The effect of siRNA/siLNA treatment was evaluated by comparing gene expression of saline treated animals with that of siRNA and siLNA treated animals respectively.

ORGANISM(S): Homo sapiens

SUBMITTER: Olaf Mook

PROVIDER: E-GEOD-6406 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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2010-05-31 | E-GEOD-16243 | biostudies-arrayexpress

Analysis of cardiac gene expression in response to isoproterenol-induced heart failure (part 2)

2010-05-31 | E-GEOD-16246 | biostudies-arrayexpress

siRna

Project description:siRna Transcriptome or Gene expression

| PRJNA308857 | ENA

Off-target effects of siRNA specific for GFP.

Project description: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.

| S-EPMC2443166 | biostudies-literature

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