Isolation and analysis of mRNAs from specific cell types of plants by ribosome immunopurification.

Angelika Mustroph, M Eugenia Zanetti, Thomas Girke, Julia Bailey-Serres

Department of Plant Physiology, University of Bayreuth, Bayreuth, Germany. angelika.mustroph@uni-bayreuth.de

Methods in molecular biology (Clifton, N.J.) 2013

filter terms:

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.

Citation

Angelika Mustroph, M Eugenia Zanetti, Thomas Girke, Julia Bailey-Serres. Isolation and analysis of mRNAs from specific cell types of plants by ribosome immunopurification. Methods in molecular biology (Clifton, N.J.). 2013;959:277-302