Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays
Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays
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DOI：
10.1038/nmeth935
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发表时间：
2006-10-01
期刊：
影响因子：
48
通讯作者：
Mathey-Prevot, Bernard
中科院分区：
文献类型：
--
作者：
Kulkarni, Meghana M.;Booker, Matthew;Mathey-Prevot, Bernard
为了评估在果蝇RNAi筛选中心（DRSC）进行的高通量RNA干扰（RNAi）筛选中使用的长dsRNAs的特异性，我们在我们的设施完成的30个全基因组筛选中对其活性进行了全球分析。值得注意的是，我们的分析预测，含有z19核苷酸完美匹配的dsRNAs在计算机上识别出的非预期靶标可能会导致脱靶效应引起的显着假阳性错误率。我们通过实验证实，dsRNA中的这些序列会导致假阳性，并导致交叉杂交转录物的有效敲低，这对基于每个基因使用单个dsRNA来解释结果提出了警告。虽然假阳性错误的所有原因仍有待确定，但我们建议一些简单的指导方针，以帮助确保从RNAi高通量筛选中获得高质量的信息。
To evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing Z19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.