Skip to main content
Disciplines
Automotive Business IT + Informatics Construction + Real Estate Electrical Engineering + Electronics Energy + Sustainability Insurance + Risk Finance + Banking Management + Leadership Marketing + Sales Mechanical Engineering + Materials
Events
DE
EN
Books
Journals
Topic Page
Marketing
Start single access now
Access for companies
EXTENDED SEARCH
Log in
Top
Published in:
Cover of the book

2015 | Online First | Chapter

Analysis of mRNA Translation Rate in Mouse Embryonic Stem Cells

Authors : Anisa B. Rahim, Leah A. Vardy

Published in: Methods in Molecular Biology™

Publisher: Springer New York

Login to get access

Activate our intelligent search to find suitable subject content or patents.

search-config
loading …

Abstract

Regulation of gene expression is essential to enable embryonic stem cells (ESCs) to either self-renew or to differentiate. Translational regulation of mRNA plays a major role in regulating gene expression and has been shown to be important for ESC differentiation. Sucrose gradients can be used to separate mRNAs based on the number of associated ribosomes and this can be used as a readout of the rate of translation. Following centrifugation through a sucrose gradient, mRNAs can be recovered, purified, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine their ribosomal load in different cell states. Here, we describe how to differentiate mouse ESCs to Neural Precursor Cells (NPCs) and analyze the rate of translation of individual mRNAs by qRT-PCR following polysome fractionation.
Literature
1.
Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147(4):789–802PubMedCentralCrossRefPubMed
2.
Sampath P, Pritchard DK, Pabon L et al (2008) A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation. Cell Stem Cell 2(5):448–460CrossRefPubMed
3.
Zhang D, Zhao T, Ang HS et al (2012) AMD1 is essential for ESC self-renewal and is translationally down-regulated on differentiation to neural precursor cells. Genes Dev 26(5):461–473PubMedCentralCrossRefPubMed
4.
5.
Hershey JW, Sonenberg N, Mathews MB (2012) Principles of translational control: an overview. Cold Spring Harb Perspect Biol 4:12CrossRef
6.
Kishore S, Luber S, Zavolan M (2010) Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression. Brief Funct Genomics 9(5–6):391–404PubMedCentralCrossRefPubMed
7.
Ingolia NT, Ghaemmaghami S, Newman JR et al (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924):218–223PubMedCentralCrossRefPubMed
8.
Ying QL, Stavridis M, Griffiths D et al (2003) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21(2):183–186CrossRefPubMed
9.
Ying QL, Smith AG (2003) Defined conditions for neural commitment and differentiation. Methods Enzymol 365:327–341CrossRefPubMed
Metadata
Title
Analysis of mRNA Translation Rate in Mouse Embryonic Stem Cells
Authors
Anisa B. Rahim
Leah A. Vardy
Copyright Year
2015
Publisher
Springer New York
DOI
https://doi.org/10.1007/7651_2015_233