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Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells

  • Ana M. V. Guedes
  • Domingos Henrique
  • Elsa Abranches
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1516)

Abstract

Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.

In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

Keywords

Stem cells Pluripotency Heterogeneity Transcription Single-molecule FISH Stochastic gene expression 

Notes

Acknowledgments

This work was supported by Fundação para a Ciência e Tecnologia, Portugal [SFRH/ BPD/78313/2011 to E.A., SFRH/BD/80191/2011 to A.M.V.G. and PTDC/SAUOBD/100664/2008].

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ana M. V. Guedes
    • 1
    • 2
  • Domingos Henrique
    • 1
    • 2
  • Elsa Abranches
    • 1
    • 2
  1. 1.DHenrique Lab, Instituto de Medicina Molecular, Faculdade de MedicinaUniversidade de LisboaLisbonPortugal
  2. 2.Instituto de Histologia e Biologia do DesenvolvimentoLisbonPortugal

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