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Myogenesis pp 229-243 | Cite as

A siRNA Mediated Screen During C2C12 Myogenesis

  • Alexis Parenté
  • Luce Pèrié
  • Laetitia Magnol
  • Khaled Bouhouche
  • Véronique BlanquetEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1889)

Abstract

Myogenesis is a multistep process taking place during pre- and postnatal stages for muscle formation, growth, and regeneration. It is a highly regulated process involving many molecular factors which act during myoblast proliferation and differentiation. To provide new insights into the molecular mechanisms and interactions behind the regulation of these different steps, RNA interference is an efficient methodology to implement. We developed a high-throughput siRNA screen in C2C12 murine myoblast cells for identification of genes relevant to signaling pathways controlling muscle growth. The proposed protocol is based on (1) the analyses of a maximum number of cells/myotubes to detect and quantify both clear and subtle phenotypes during proliferation/fusion cells and (2) the use of two cellular fluorescent markers, DAPI and myosin, decorating nuclei and myotubes respectively. Four phenotypic criteria were quantitatively assessed: cellular density, myotubes quantity, fusion index, and size and morphology of myotubes.

Key words

siRNA Functional screen Myogenesis C2C12 Quantitative-imaging analysis Proliferation Differentiation Immunostaining 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alexis Parenté
    • 1
  • Luce Pèrié
    • 1
  • Laetitia Magnol
    • 1
  • Khaled Bouhouche
    • 1
  • Véronique Blanquet
    • 1
    Email author
  1. 1.INRA, PEIRENE EA7500, USC1061 GAMAA, Université de LimogesLimogesFrance

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