期刊
METHODS
卷 187, 期 -, 页码 68-76出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2020.04.009
关键词
Super resolution microscopy; STED; lncRNA; Nuclear architecture; Nuclear domains
资金
- Spanish Ministry of Health Plan Nacional de I + D + I, ISCIII, FEDER [FIS PI09/02444, PI12/00511, PI15/01763]
- Agencia de Gestio d'Ajuts Universitaris i de Recerca [2014-SGR-1269]
- Instituto de Salud Carlos III [PIE16-00011]
- Fundacio Olga Torres grant
Super resolution microscopy and FISH techniques were used to visualize the precise location and interactions of a novel lncRNA TNBL in the nucleus, providing insights into its functionality. Optimized protocols can be employed for single-cell imaging of complex nuclear RNA-protein structures, aiding in understanding spatial arrangements within cells.
Super resolution microscopy has changed our capability to visualize and understand spatial arrangements of RNA- and protein-containing domains in individual cells. In a previous study, we described a novel lncRNA, Tumor-associated NBL2 transcript (TNBL), which originates from a primate specific macrosatellite repeat. We aimed to describe several aspects of TNBL lncRNA, with one focus being pinpointing its precise location in the nucleus, as well as visualizing its interactions with proteins to deduce its functionality. Using a combination of STimulated Emission Depletion (STED) super resolution microscopy, single molecule RNA (smRNA) FISH against TNBL, and immunofluorescence against SAM68 perinucleolar body, we resolved the spatial complexity of the interaction between TNBL aggregates and SAM68 bodies at the perinucleolar region. Here, we describe protocols for a step-by-step optimized smRNA FISH/IF and STED imaging, detailing parameter settings, and three-dimensional data analysis of spatial positioning of subnuclear structures. These protocols can be employed for single-cell imaging of complex nuclear RNA-protein structures.
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