Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading

被引：145
作者：
Gagnon, Keith T. [1 ]
Li, Liande [1 ]
Janowski, Bethany A. [1 ]
Corey, David R. [1 ,2 ]
机构：
[1] Univ Texas SW Med Ctr Dallas, Dept Pharmacol, Dallas, TX 75390 USA
[2] Univ Texas SW Med Ctr Dallas, Dept Biochem, Dallas, TX 75390 USA
来源：
NATURE PROTOCOLS | 2014年 / 9卷 / 09期
基金：
美国国家卫生研究院;
关键词：
DOUBLE-STRANDED-RNA; GENE-EXPRESSION; PROMOTER; LOCALIZATION; INVOLVEMENT; ACTIVATION; PROTEINS; AGO2; DNA;
D O I：
10.1038/nprot.2014.135
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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 include a method for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. This protocol facilitates the characterization of nuclear RNAi, and it 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.
引用
收藏
页码：2045 / 2060
页数：16
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