Single-Molecule Live-Cell Visualization of Pre-mRNA Splicing
Microscopy protocols that allow live-cell imaging of molecules and subcellular components tagged with fluorescent conjugates are indispensable in modern biological research. A breakthrough was recently introduced by the development of genetically encoded fluorescent tags that combined with fluorescence-based microscopic approaches of increasingly higher spatial and temporal resolution made it possible to detect single protein and nucleic acid molecules inside living cells. Here, we describe an approach to visualize single nascent pre-mRNA molecules and to measure in real time the dynamics of intron synthesis and excision.
Key wordsSingle molecule Live-cell imaging Spinning disk confocal microscopy Splicing Pre-mRNA
We gratefully acknowledge Tomas Kirchhausen and members of the Kirchhausen lab for advice and support during the development of this protocol. This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/118180/2010; SFRH/BPD/66611/2009), and the Harvard Medical School-Portugal Program in Translational Research and Information.
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