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

Keith T. Gagnon, Liande Li, Bethany A. Janowski, David R. Corey

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2045-2060
Number of pages16
JournalNature Protocols
Volume9
Issue number9
DOIs
StatePublished - 2014

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Cell Fractionation
Nuclear RNA
Fractionation
RNA Interference
Cells
Cell Nucleus
Argonaute Proteins
Nuclear Proteins
Gene expression
Chromatin
Contamination
RNA
Cultured Cells
Cytoplasm
Gene Expression

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. / Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

In: Nature Protocols, Vol. 9, No. 9, 2014, p. 2045-2060.

Research output: Contribution to journalArticle

Gagnon, Keith T. ; Li, Liande ; Janowski, Bethany A. ; Corey, David R. / Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. In: Nature Protocols. 2014 ; Vol. 9, No. 9. pp. 2045-2060.
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