Analysis of global RNA synthesis at the single cell level following hypoxia.
Analysis of global RNA synthesis at the single cell level following hypoxia.
复制标题
DOI：
10.3791/51420
复制
发表时间：
2014-05-13
期刊：
影响因子：
--
通讯作者：
Rocha S
中科院分区：
文献类型：
--
作者：
Biddlestone J;Druker J;Shmakova A;Ferguson G;Swedlow JR;Rocha S
缺氧或氧气利用率降低涉及许多生理和病理过程。在分子水平上，细胞启动特定的转录程序，以产生适当且协调的细胞反应。该细胞拥有多种氧传感器酶，需要分子氧作为其活性的辅助因子。这些酶的范围从脯氨酰羟化酶到组蛋白去甲基化酶。大多数分析细胞对缺氧反应的研究都是基于细胞群和平均研究，因此很少进行缺氧细胞的单细胞分析。在这里，我们描述了一种在缺氧情况下在单细胞水平上分析全局 RNA 合成的方法，方法是在控氧工作站中使用 Click-iT RNA 成像试剂盒，然后进行显微镜分析和定量。 使用暴露于缺氧不同时间长度的癌细胞，对每个细胞中的 RNA 进行标记和测量。该分析可以可视化低氧应激后整体 RNA 合成的时间和细胞间变化。
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.
登录
查看更多内容
影响因子：
15
作者：
Wang, Q;Chan, TR;Finn, MG
通讯作者：
Finn, MG
影响因子：
20.3
作者：
Schoedel, Johannes;Oikonomopoulos, Spyros;Mole, David R.
通讯作者：
Mole, David R.
DOI：
10.1073/pnas.0808480105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Jao, Cindy Y.;Salic, Adrian
通讯作者：
Salic, Adrian
影响因子：
5.3
作者：
Culver, Carolyn;Sundqvist, Anders;Rocha, Sonia
通讯作者：
Rocha, Sonia
影响因子：
48
作者：
Allan, Chris;Burel, Jean-Marie;Moore, Josh;Blackburn, Colin;Linkert, Melissa;Loynton, Scott;MacDonald, Donald;Moore, William J.;Neves, Carlos;Patterson, Andrew;Porter, Michael;Tarkowska, Aleksandra;Loranger, Brian;Avondo, Jerome;Lagerstedt, Ingvar;Lianas, Luca;Leo, Simone;Hands, Katherine;Hay, Ron T.;Patwardhan, Ardan;Best, Christoph;Kleywegt, Gerard J.;Zanetti, Gianluigi;Swedlow, Jason R.
通讯作者：
Swedlow, Jason R.