title: Analysis of global RNA synthesis at the single cell level following hypoxia.
abstract: 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.
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK.
authors.forename: John
authors.initials: J
authors.lastname: Biddlestone
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK.
authors.forename: Jimena
authors.initials: J
authors.lastname: Druker
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK.
authors.forename: Alena
authors.initials: A
authors.lastname: Shmakova
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK.
authors.forename: Gus
authors.initials: G
authors.lastname: Ferguson
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Swedlow
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK; s.rocha@dundee.ac.uk.
authors.forename: Sonia
authors.initials: S
authors.lastname: Rocha
mesh_terms: D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods; D012313:RNA / Q000096:biosynthesis*; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D016328:NF-kappa B; D012313:RNA