C911: A bench-level control for sequence specific siRNA off-target effects.
C911: A bench-level control for sequence specific siRNA off-target effects.
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C911：用于序列特定siRNA脱靶效应的基准级控制。
DOI：
10.1371/journal.pone.0051942
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Martin S
中科院分区：
文献类型：
--
作者：
Buehler E;Chen YC;Martin S
小干扰RNA（siRNA）已成为下调mRNA的普遍实验工具。不幸的是，脱靶效应是SiRNA实验中假阳性的重要来源，并且之前尚未确定对其的有效控制。我们介绍了两种基于将siRNA中间的碱基改变为它们的互补碱基的用于阴性对照的错配siRNA设计方法。为了测试这些对照，从在表达简单的组成型表达的荧光素酶报告基因的细胞系中进行的全基因组筛选中鉴定了20种高活性siRNA（10种真阳性和10种假阳性）的测试组。然后为这20种siRNA中的每一种合成三种对照，前两种使用所提出的错配设计方法，第三种是序列的简单随机排列（乱序siRNA）。当在原始测定中测试时，与原始siRNA相比，乱序siRNA显示出显著降低的活性，无论它们是否被鉴定为真阳性或假阳性，表明它们作为实验对照几乎没有效用。相比之下，其中一种被称为C911的错配设计方法，因为siRNA的碱基9到11被它们的互补序列取代，能够完全区分这两组。当检测C911错配对照时，由于脱靶效应导致的假阳性保持了大部分活性，而当检测C911错配时，表型由于中靶效应导致的真阳性失去了大部分或全部活性。控制siRNA区分真阳性和假阳性的能力，如果被广泛采用，可以减少文献中报告的错误结果，并节省花费在昂贵的后续实验上的研究资金。
Small interfering RNAs (siRNAs) have become a ubiquitous experimental tool for down-regulating mRNAs. Unfortunately, off-target effects are a significant source of false positives in siRNA experiments and an effective control for them has not previously been identified. We introduce two methods of mismatched siRNA design for negative controls based on changing bases in the middle of the siRNA to their complement bases. To test these controls, a test set of 20 highly active siRNAs (10 true positives and 10 false positives) was identified from a genome-wide screen performed in a cell-line expressing a simple, constitutively expressed luciferase reporter. Three controls were then synthesized for each of these 20 siRNAs, the first two using the proposed mismatch design methods and the third being a simple random permutation of the sequence (scrambled siRNA). When tested in the original assay, the scrambled siRNAs showed significantly reduced activity in comparison to the original siRNAs, regardless of whether they had been identified as true or false positives, indicating that they have little utility as experimental controls. In contrast, one of the proposed mismatch design methods, dubbed C911 because bases 9 through 11 of the siRNA are replaced with their complement, was able to completely distinguish between the two groups. False positives due to off-target effects maintained most of their activity when the C911 mismatch control was tested, whereas true positives whose phenotype was due to on-target effects lost most or all of their activity when the C911 mismatch was tested. The ability of control siRNAs to distinguish between true and false positives, if widely adopted, could reduce erroneous results being reported in the literature and save research dollars spent on expensive follow-up experiments.
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DOI：
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