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Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing

Author:
Wolfe, Michael B., Goldstrohm, Aaron C., Freddolino, Peter L.
Source:
Methods 2019 v.155 pp. 88-103
ISSN:
1046-2023
Subject:
high-throughput nucleotide sequencing, messenger RNA, metabolism, transcription (genetics), transcriptome
Abstract:
Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.
Agid:
6252230