Isolation and analysis of mrnas from specific cell types of plants by ribosome immunopuri fication

Abstract

Multiple ribosomes assemble onto an individual mRNA to form a polyribosome (polysome) complex. The epitope tagging of speci fi c ribosomal proteins can enable the immunopuri fi cation of polysomes from crude cell extracts derived from cryopreserved tissue samples. Through expression of the epitope-tagged ribosomal protein in cell-type and regional speci fi c domains of Arabidopsis thaliana and other organisms it is feasible to quantitatively assess the mRNAs that are associated with ribosomes with cell-speci fi c 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 speci fi c domains of expression, the immunopuri fi cation of ribosomes, and bioinformatic analyses of the resultant datasets obtained by microarray pro fi ling. This methodology provides researchers with the opportunity to assess rapid changes at the organ, tissue, regional or cell-type speci fi c level of mRNAs that are associated with ribosomes and therefore engaged in translation. © Springer Science+Business Media New York 2013.