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Related Experiment Video

Updated: May 20, 2025

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
10:00

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Published on: October 28, 2014

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Protocol to perform polysome profiling in primary differentiating murine adipocytes.

Mirian Krystel De Siqueira1, Zaynab Nouhi2, Yutian Zhao1

  • 1Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

STAR Protocols
|May 2, 2025
PubMed
Summary

We developed a polysome profiling protocol to study how protein synthesis is regulated in differentiating mouse fat cells. This method helps understand gene expression changes in metabolic diseases like obesity.

Keywords:
Cell BiologyGene ExpressionMetabolismRNA-seq

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Last Updated: May 20, 2025

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Adipogenesis involves complex gene regulation.
  • Understanding translational control is crucial for metabolic research.
  • Existing methods may not fully capture dynamic changes in protein synthesis during adipocyte differentiation.

Purpose of the Study:

  • To present a detailed protocol for polysome profiling in differentiating mouse adipocytes.
  • To enable the analysis of actively translating messenger RNAs (mRNAs).
  • To investigate translational gene regulation in the context of adipogenesis.

Main Methods:

  • Isolation of polysome-bound mRNA from mouse stromal vascular fraction.
  • Preparation of cell lysates.
  • Ultracentrifugation through sucrose gradients.
  • RNA sequencing and motif enrichment analysis.

Main Results:

  • The protocol successfully isolates polysome-bound mRNA.
  • Enables identification of actively translated genes during adipocyte differentiation.
  • Provides insights into translational control mechanisms.

Conclusions:

  • This protocol offers a robust method for studying translational regulation in adipocytes.
  • Facilitates research into metabolic diseases, obesity, and related cellular phenotypes.
  • Links translational control to cellular function and metabolic status.