IBBM   21076
INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
capítulos de libros
Título:
Isolation and Analysis of mRNAs from specific Cell Types of Plants by Ribosome Immunopurification
Autor/es:
ANGLEIKA MUSTROPH; MAR√ćA EUGENIA ZANETTI; THOMAS GIRKE; JULIA BAILEY-SERRES
Libro:
Plant organogenesis: Methods and protocols, Methods in Molecular Biology
Editorial:
Springer Science +Business Media New York
Referencias:
Lugar: New York; Año: 2012; p. 277 - 302
Resumen:
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. 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. 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. 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. 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. 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.