Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading.
Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading.
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DOI：
10.1038/nprot.2014.135
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发表时间：
2014-09
期刊：
影响因子：
14.8
通讯作者：
Corey, David R.
中科院分区：
文献类型：
--
作者：
Gagnon, Keith T.;Li, Liande;Janowski, Bethany A.;Corey, David R.
RNA干扰（RNAi）以其调节哺乳动物细胞质中基因表达的能力而众所周知。然而，在哺乳动物细胞核中，RNAi的影响仍然存在争议。一个关键的技术障碍是缺乏用于分离和分析细胞核的优化方案。在这里，我们描述了一个简化的协议，从培养的细胞，包括获得核质和染色质组分和去除细胞质污染的方法分离细胞核。然后可以使用细胞级分来检测细胞核中RNAi因子的存在和活性。我们提出了一个研究RNAi早期步骤的方案，Argonaute蛋白负载小RNA，这是通过我们改进的提取物制备实现的。这些方案有助于核RNAi的表征，并可应用于其他核蛋白和途径的分析。从细胞分离到Argonaute加载结果分析，该方案需要4-6天才能完成。
RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete.
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