Tschuch Cordula, Schulz Angela, Pscherer Armin, Werft Wiebke, Benner Axel, Hotz-Wagenblatt Agnes, Barrionuevo Leticia Serra, Lichter Peter, Mertens Daniel
Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany.
BMC Mol Biol. 2008 Jun 24;9:60. doi: 10.1186/1471-2199-9-60.
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.
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.
通过RNA干扰进行基因敲低是在实验和治疗环境中沉默基因表达的一种高效方法。然而，这种广泛使用的方法存在严重缺陷，尤其是在RNA干扰分子的特异性方面。
我们测试了最广泛使用的针对绿色荧光蛋白（GFP）的对照小干扰RNA（siRNA）的脱靶效应，发现它除了使GFP沉默外，还会使一组内源性靶基因失调。脱靶效应取决于转染的GFP siRNA的量，并且在多种细胞系中都能检测到。由于特异性针对GFP的相应siRNA分子被广泛用作RNA干扰实验的阴性对照，我们通过全基因组表达谱研究了该分子的完整脱靶基因集。检测到的受调控mRNA具有与小至8个碱基对大小的siRNA同源的靶序列。然而，我们发现靶基因的3'非翻译区（3'UTR）不存在序列同源性限制。
我们可以证明，即使是没有生理靶标的siRNA在哺乳动物细胞中也具有序列特异性脱靶效应。此外，我们的分析确定了受常用作阴性对照且针对GFP的siRNA影响的脱靶基因。由于脱靶效应几乎无法避免，最好的策略是识别假阳性并将其从结果中排除。为此，我们提供了由常用的GFP siRNA失调的假阳性基因集，作为未来siRNA实验的参考资源。
