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.
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Acknowledgment
This work was partly supported by Starting-up grant of Henan Normal University (5101049470095) and Program for Innovative Research Team in Science and Technology in University of Henan Province (17IRTSTHN017) to YFL. The work in RS laboratory was supported by the Oklahoma Agricultural Experiment Station.
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Li, YF., Mahalingam, R., Sunkar, R. (2017). Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants. In: Sunkar, R. (eds) Plant Stress Tolerance. Methods in Molecular Biology, vol 1631. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7136-7_9
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DOI: https://doi.org/10.1007/978-1-4939-7136-7_9
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