Kulkarni Meghana M, Booker Matthew, Silver Serena J, Friedman Adam, Hong Pengyu, Perrimon Norbert, Mathey-Prevot Bernard
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Nat Methods. 2006 Oct;3(10):833-8. doi: 10.1038/nmeth935.
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 > or = 19-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.
为评估在果蝇RNA干扰（RNAi）筛选中心（DRSC）进行的高通量RNA干扰（RNAi）筛选中使用的长双链RNA（dsRNA）的特异性，我们对在我们设施中完成的30个全基因组筛选中它们的活性进行了全面分析。值得注意的是，我们的分析预测，在计算机模拟中鉴定出与非预期靶标具有≥19个核苷酸完全匹配的dsRNA，可能会导致由脱靶效应引起的显著假阳性错误率。我们通过实验证实，dsRNA中的此类序列会导致假阳性，并导致交叉杂交转录本的有效敲低，这对基于每个基因使用单个dsRNA来解释结果提出了警示。尽管对假阳性错误的所有原因的全面理解仍有待确定，但我们建议了一些简单的指导原则，以帮助确保从RNAi高通量筛选中获得高质量信息。
