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

  • Angelika MustrophEmail author
  • M. Eugenia Zanetti
  • Thomas Girke
  • Julia Bailey-Serres
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Ribosome immunopurification Polysomes Translatome Cell-specific gene expression Microarray Differential expression analysis 

Notes

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.).

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Angelika Mustroph
    • 1
    Email author
  • M. Eugenia Zanetti
    • 2
    • 3
    • 4
  • Thomas Girke
    • 5
  • Julia Bailey-Serres
    • 3
    • 2
  1. 1.Department of Plant PhysiologyUniversity of BayreuthBayreuthGermany
  2. 2.Department of Botany and Plant SciencesUniversity of CaliforniaRiversideUSA
  3. 3.Center for Plant Cell BiologyUniversity of CaliforniaRiversideUSA
  4. 4.Facultad de Ciencias Exactas, Instituto de Biotecnología y Biología MolecularUniversidad Nacional de La PlataLa PlataArgentina
  5. 5.Department of Botany and Plant Sciences, Center for Plant Cell Biology (CEPCEB), Institute of Integrative Genome Biology (IIGB)University of CaliforniaRiversideUSA

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