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Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzyme...

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Autores principales: Biddlestone, John, Druker, Jimena, Shmakova, Alena, Ferguson, Gus, Swedlow, Jason R., Rocha, Sonia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184815/
https://www.ncbi.nlm.nih.gov/pubmed/24895009
http://dx.doi.org/10.3791/51420
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author Biddlestone, John
Druker, Jimena
Shmakova, Alena
Ferguson, Gus
Swedlow, Jason R.
Rocha, Sonia
author_facet Biddlestone, John
Druker, Jimena
Shmakova, Alena
Ferguson, Gus
Swedlow, Jason R.
Rocha, Sonia
author_sort Biddlestone, John
collection PubMed
description Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.
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spelling pubmed-41848152014-10-06 Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia Biddlestone, John Druker, Jimena Shmakova, Alena Ferguson, Gus Swedlow, Jason R. Rocha, Sonia J Vis Exp Cellular Biology Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress. MyJove Corporation 2014-05-13 /pmc/articles/PMC4184815/ /pubmed/24895009 http://dx.doi.org/10.3791/51420 Text en Copyright © 2014, Journal of Visualized Experiments http://creativecommons.org/licenses/by/3.0/us/ This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. To view a copy of this license, visithttp://creativecommons.org/licenses/by/3.0/us/
spellingShingle Cellular Biology
Biddlestone, John
Druker, Jimena
Shmakova, Alena
Ferguson, Gus
Swedlow, Jason R.
Rocha, Sonia
Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title_full Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title_fullStr Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title_full_unstemmed Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title_short Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
title_sort analysis of global rna synthesis at the single cell level following hypoxia
topic Cellular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184815/
https://www.ncbi.nlm.nih.gov/pubmed/24895009
http://dx.doi.org/10.3791/51420
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