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Isolation and Analysis of mRNAs from Specific Cell Types of Plants by Ribosome Immunopurification

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Plant Organogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 959))

Abstract

Multiple ribosomes assemble onto an individual mRNA to form a polyribosome (polysome) complex. The epitope tagging of specific ribosomal proteins can enable the immunopurification of polysomes from crude cell extracts derived from cryopreserved tissue samples. Through expression of the epitope-tagged ribosomal protein in cell-type and regional specific domains of Arabidopsis thaliana and other organisms it is feasible to quantitatively assess the mRNAs that are associated with ribosomes with cell-specific resolution. Here we present detailed methods for development of transgenics that express a FLAG-tagged version of ribosomal protein L18 (RPL18) under the direction of individual promoters with specific domains of expression, the immunopurification of ribosomes, and bioinformatic analyses of the resultant datasets obtained by microarray profiling. This methodology provides researchers with the opportunity to assess rapid changes at the organ, tissue, regional or cell-type specific level of mRNAs that are associated with ribosomes and therefore engaged in translation.

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Acknowledgments

We thank all of the individuals who have contributed to the development of polysome isolation and analyses methods in our group over the years. This work was supported by the U.S. National Science Foundation (DBI 0211857, IBN-0420152, and IOS-0750811 to J. B.-S. and 2010-0820842 and ABI-0957099 to T. G.).

Author information

Authors and Affiliations

  1. Department of Plant Physiology, University of Bayreuth, Bayreuth, Germany

    Angelika Mustroph

  2. Department of Botany and Plant Sciences, University of California, Riverside, CA, USA

    M. Eugenia Zanetti & Julia Bailey-Serres

  3. Center for Plant Cell Biology, University of California, Riverside, CA, USA

    M. Eugenia Zanetti & Julia Bailey-Serres

  4. Facultad de Ciencias Exactas, Instituto de Biotecnología y Biología Molecular, Universidad Nacional de La Plata, La Plata, Argentina

    M. Eugenia Zanetti

  5. Department of Botany and Plant Sciences, Center for Plant Cell Biology (CEPCEB), Institute of Integrative Genome Biology (IIGB), University of California, Riverside, CA, USA

    Thomas Girke

Authors
  1. Angelika Mustroph
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  2. M. Eugenia Zanetti
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  3. Thomas Girke
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  4. Julia Bailey-Serres
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Corresponding author

Correspondence to Angelika Mustroph .

Editor information

Editors and Affiliations

  1. School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, United Kingdom

    Ive De Smet

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Mustroph, A., Zanetti, M.E., Girke, T., Bailey-Serres, J. (2013). Isolation and Analysis of mRNAs from Specific Cell Types of Plants by Ribosome Immunopurification. In: De Smet, I. (eds) Plant Organogenesis. Methods in Molecular Biology, vol 959. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-221-6_19

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  • DOI: https://doi.org/10.1007/978-1-62703-221-6_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-220-9

  • Online ISBN: 978-1-62703-221-6

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