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Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay

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mRNA Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1648))

Abstract

The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.

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Acknowledgments

Research in the Hertel laboratory is supported by NIH (GM062287 and GM110244). Maliheh Movassat and Hossein Shenasa contributed equally to this work.

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Author notes

  1. Maliheh Movassat and Hossein Shenasa contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA

    Maliheh Movassat, Hossein Shenasa & Klemens J. Hertel

Authors
  1. Maliheh Movassat
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  2. Hossein Shenasa
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  3. Klemens J. Hertel
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Corresponding author

Correspondence to Klemens J. Hertel .

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Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA

    Yongsheng Shi

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Movassat, M., Shenasa, H., Hertel, K.J. (2017). Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay. In: Shi, Y. (eds) mRNA Processing. Methods in Molecular Biology, vol 1648. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7204-3_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7204-3_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7203-6

  • Online ISBN: 978-1-4939-7204-3

  • eBook Packages: Springer Protocols

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