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ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Publications at this Location » Publication #339220

Research Project: Improvement of Barley Seed Quality Through Molecular and Functional Genomic Gene Expression

Location: Cereal Crops Research

Title: Isolation of polysomal RNA for analyzing stress-responsive genes regulated at the translational level in plants

item LI, YONG-FANG - Henan Normal University
item Mahalingam, Ramamurthy
item SUNKAR, RAMANJULU - Oklahoma State University

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 3/24/2017
Publication Date: 8/30/2017
Citation: Li, Y., Mahalingam, R., Sunkar, R. 2017. Isolation of polysomal RNA for analyzing stress-responsive genes regulated at the translational level in plants. In: Ramanjulu Sunkar, editor. Plant stress tolerance: Methods and Protocols, 2nd edition. Humana Press. p. 151-161.

Interpretive Summary: In order to identify genes that are involved in response to stresses, comparisons of RNA between plants growing under controlled conditions are compared with plants growing under stressed conditions. However, changes in gene expression levels does not correlate well with changes at the protein levels. This indicates there are other levels of regulation between transcription and protein synthesis. Messenger RNA associated with ribosomes represent what is called polysomal RNA. These polysomal RNA in fact represents the nascent proteome and can provide a snapshot of the genes that are going to be translated or are in the process of translation. In this chapter, we describe a step-by-step protocol for isolating polysomal RNA from plant tissues that can be useful for analyzing gene expression via RNA-seq, qPCR or microarray technologies.

Technical Abstract: Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including northern blot analysis, qRT-PCR, RNase protection assay and microarray based gene expression profiling.