Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells
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.
KeywordsStem cells Pluripotency Heterogeneity Transcription Single-molecule FISH Stochastic gene expression
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].
- 28.Core Team RCTR (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
- 29.Pezzarossa A, Guedes A, Henrique D, Abranches E (2015) Imaging pluripotency time-lapse analysis of mouse embryonic stem cells. Embryonic Stem Cell Protocols, Volume 1341 of the series Methods in Molecular Biology pp 87–100Google Scholar
- 31.Raj A, Tyagi S (2009) Imaging individual mrna molecules using multiple singly labeled probes, US Patent App. 13/062. http://www.google.com/patents/US20120129165