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

John Biddlestone, Jimena Druker, Alena Shmakova, Gus Ferguson, Jason R. Swedlow, Sonia Rocha (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
164 Downloads (Pure)

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.
Original languageEnglish
Article numbere51420
Number of pages7
JournalJoVE: Journal of Visualized Experiments
Issue number87
DOIs
Publication statusPublished - 13 May 2014

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