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Single-Molecule Live-Cell Visualization of Pre-mRNA Splicing

  • Robert M. Martin
  • José Rino
  • Ana C. de Jesus
  • Maria Carmo-FonsecaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1358)

Abstract

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 words

Single molecule Live-cell imaging Spinning disk confocal microscopy Splicing Pre-mRNA 

Notes

Acknowledgements

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Robert M. Martin
    • 1
  • José Rino
    • 1
  • Ana C. de Jesus
    • 1
  • Maria Carmo-Fonseca
    • 1
    Email author
  1. 1.Faculdade de Medicina, Instituto de Medicina MolecularUniversidade de LisboaLisbonPortugal

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