Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification

Xingfa Han; Laura L. Burger; David Garcia-Galiano; Suzanne M. Moenter; Martin G. Myers, Jr,; David P. Olson; Carol F. Elias

STAR Protocols (Jun 2021)

Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification

Xingfa Han,
Laura L. Burger,
David Garcia-Galiano,
Suzanne M. Moenter,
Martin G. Myers, Jr,,
David P. Olson,
Carol F. Elias

Affiliations

Xingfa Han: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Isotope Research Lab, Sichuan Agricultural University, Ya’an 625014, China
Laura L. Burger: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
David Garcia-Galiano: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
Suzanne M. Moenter: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48109, USA; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI 48109, USA
Martin G. Myers, Jr,: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48109, USA
David P. Olson: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48109, USA; Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI 48109, USA; Corresponding author
Carol F. Elias: Department of Molecular & integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI 48109, USA; Corresponding author

Journal volume & issue: Vol. 2, no. 2
p. 100589

Abstract

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Summary: Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol.For complete details on the use and execution of this protocol, please refer to Han et al. (2020).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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