Assays for direct and indirect effects of C. elegans endo-siRNAs.
Assays for direct and indirect effects of C. elegans endo-siRNAs.
复制标题
线虫内切 siRNA 的直接和间接影响的测定。
DOI：
10.1007/978-1-4939-0931-5_7
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Hunter,CraigP
中科院分区：
文献类型：
--
作者：
Shiu,PhilipK;Zhuang,JimmyJ;Hunter,CraigP
自从在C.随着秀丽隐杆线虫的发现，越来越多的内源性小RNA被发现。内源性siRNA（endo-siRNA）在过去几年中已经作为调节内源基因表达的基本上独立的小RNA类别出现，其机制不同于piRNA和miRNA的机制。定量这些小RNA及其对靶RNA的影响是分析RNAi的有力工具;然而，由于小RNA的尺寸小且丰度相对较低，因此检测小RNA可能很困难。在这里，我们描述了用于直接检测大量内siRNA水平的新型Firecraft测定，以及用于检测内siRNA对基因靶标的影响的优化qPCR方法。有趣的是，endo-siRNA的丢失经常导致增强的实验性RNAi。因此，我们还提出了一种优化的方法来评估endo-siRNA对实验RNAi效率的间接影响。
Ever since the discovery of the first microRNAs inC. elegans, increasing numbers of endogenous small RNAs have been discovered. Endogenous siRNAs (endo-siRNAs) have emerged in the last few years as a largely independent class of small RNAs that regulate endogenous gene expression, with mechanisms distinct from those of piRNAs and miRNAs. Quantification of these small RNAs and their effect on target RNAs is a powerful tool for the analysis of RNAi; however, detection of small RNAs can be difficult due to their small size and relatively low abundance. Here, we describe the novel FirePlex assay for directly detecting endo-siRNA levels in bulk, as well as an optimized qPCR method for detecting the effect of endo-siRNAs on gene targets. Intriguingly, the loss of endo-siRNAs frequently results in enhanced experimental RNAi. Thus, we also present an optimized method to assess the indirect impact of endo-siRNAs on experimental RNAi efficiency.