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The choice of resuspension medium for isolated rat liver nuclei: Effects on nuclear morphology andin vitro transcription

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Abstract

Standard protocols forin vitro transcription assay (nuclear run-off) include 10–40% (v/v) glycerol (of various ionic strength) in the medium used for resuspension/storage of the isolated nuclei. In the present work the morphological and functional properties of nuclei isolated from rat liver have been studied as a function of the content of glycerol, sucrose and inorganic ions (K+ and Mg2+) in the resuspension medium.

In contrast to earlier reports, glycerol was found not to be essential to maintain morphological integrity and RNA polymerase activity in frozen/stored nuclei. Nuclear pellets, resuspended and stored in isoosmotic sucrose media, were found to give morphologically intact and transcriptionally active nuclei. Furthermore, these nuclei displayed a higher specific hybridization signal for the differentially expressed genes encoding peroxisomal β-oxidation enzymes, relative to the total RNA synthesis, than nuclei resuspended and stored in a hyperosmotic glycerol-containing medium.

The concentrations of inorganic ions were also found to affect nuclear morphology. Flow cytometry indicated DNA leakage from nuclei at insufficient concentrations of K+ and Mg2+, and high ionic strength favoured aggregation and disintegration of nuclei.

Our findings indicate that quantitative results from nuclear run-off experiments should be interpreted with caution until the process of transcription in isolated nuclei is better understood.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, N-5009, Bergen, Norway

    Roger Strand & Torgeir Flatmark

  2. Department of Anatomy and Cell Biology, University of Bergen, Bergen, Norway

    Roald Bøe

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  1. Roger Strand
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  2. Roald Bøe
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Strand, R., Bøe, R. & Flatmark, T. The choice of resuspension medium for isolated rat liver nuclei: Effects on nuclear morphology andin vitro transcription. Mol Cell Biochem 139, 149–157 (1994). https://doi.org/10.1007/BF01081738

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  • DOI: https://doi.org/10.1007/BF01081738

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