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

Nature Protocols volume 9, pages 2045–2060 (2014)Cite this article

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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.

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Figure 1
Figure 2: Schematic of the in vitro Ago2 loading assay.
Figure 3: Quality assessment of subcellular fractionation.
Figure 4: In vitro Ago2 loading assay and typical results from Step 9B.

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Acknowledgements

Funding was provided by the US National Institutes of Health (1F32HD060377/KTG, GM 73042/DRC and GM85080/BAJ), the Welch Foundation (I-1244/DRC) and the Cancer Prevention and Research Institute of Texas (RP120311/BAJ).

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Authors and Affiliations

  1. Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA

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

  2. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA

    David R Corey

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  1. Keith T Gagnon
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  2. Liande Li
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  3. Bethany A Janowski
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  4. David R Corey
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Contributions

K.T.G. and L.L. designed and performed the experiments, including optimization of subcellular fractionation and development of the in vitro Argonaute loading assay. K.T.G., L.L., B.A.J. and D.R.C. all participated in the writing of this manuscript.

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Correspondence to David R Corey.

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The authors declare no competing financial interests.

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Gagnon, K., Li, L., Janowski, B. et al. Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat Protoc 9, 2045–2060 (2014). https://doi.org/10.1038/nprot.2014.135

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