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Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays

Nature Protocols volume 1, pages 3094–3100 (2006)Cite this article

Abstract

Isolation of transcriptionally active nuclei from plant tissues is a fundamental first step in many plant molecular biology protocols. Enriched nuclear fractions may be used in “run-on” assays to measure the rate of transcription for any given gene, adding additional resolution to assays of steady-state transcript accumulation such as RNA-gel blots, RT–PCR or microarrays. The protocols presented here streamline, adapt and optimize existing methods for use in Arabidopsis thaliana. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. This capability complements the immense body of steady-state transcript measurements and indirectly identifies instances where message turnover may have a critical and/or primary role in regulating gene expression levels.

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Figure 1: Fractionation by Percoll gradients.
Figure 2: Microscopic images of isolated nuclei.
Figure 3: An example of results from a nuclear run-on assay.

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Acknowledgements

We thank Scott Nicholson for critical evaluation of this protocol and Dr Ana-Lisa Paul for assistance with microscopy and imaging. This work was performed with startup funding from the University of Florida (KMF).

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Authors and Affiliations

  1. Horticultural Sciences Department and the Plant Molecular and Cellular Biology Program, University of Florida, 1301 Fifield Hall, Gainesville, 32611, Florida, USA

    Kevin M Folta

  2. Department of Biology, Laboratory for Molecular Biology, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, 60612, Illinois, USA

    Lon S Kaufman

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Correspondence to Kevin M Folta.

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Folta, K., Kaufman, L. Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat Protoc 1, 3094–3100 (2006). https://doi.org/10.1038/nprot.2006.471

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