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Study of X Chromosome Activity Status in Human Naive Pluripotent Stem Cells Using RNA-FISH

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Human Naïve Pluripotent Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2416))

Abstract

X chromosome activity is a defining attribute of naive pluripotency, with naive pluripotency being a rare context in which both X chromosomes of females are active. RNA-fluorescence in situ hybridization (RNA-FISH) is a powerful tool to determine the transcriptional status of specific genes with allelic and single-cell resolution and has been widely used in the context of X chromosome inactivation, the process ensuring dosage compensation for X-linked genes between sexes in mammals. RNA-FISH using genomic or intronic probes allows the detection of newly synthesized transcripts at the site of transcription. This technique is invaluable for appreciating the putative heterogeneity in the expression profiles within cell populations. RNA-FISH has the added advantage of allowing the visualization of gene transcription in a spatial perspective. Here, we provide a detailed protocol describing the application of RNA-FISH to detect nascent X-linked transcripts in female naive human embryonic stem cells to assess their X chromosome status, along with another complementary technique, DNA-FISH.

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Acknowledgments

We thank members of the Rougeulle laboratory for their contribution to the elaboration of the procedures described in this chapter and Céline Morey for critical inputs as well as reading of the manuscript. We thank the Microscopy Platform hosted in UMR7216, Epigenetics and Cell Fate Unit for their support. Research leading to these results has received funding from Agence Nationale pour la Recherche (ANR-14-CE10-0017) and from Ligue Nationale contre le Cancer. This study was also supported by LabEx “Who Am I?” (ANR-11-LABX-0071) and Université de Paris IdEx (ANR-18-IDEX-0001) funded by the French Government through its “Investments for the Future” program. K.M. is supported by a fellowship from the French Medical Research Foundation (FRM SPF201809006928).

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Correspondence to Claire Rougeulle .

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Mahadik, K., Rougeulle, C. (2022). Study of X Chromosome Activity Status in Human Naive Pluripotent Stem Cells Using RNA-FISH . In: Rugg-Gunn, P. (eds) Human Naïve Pluripotent Stem Cells. Methods in Molecular Biology, vol 2416. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1908-7_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1908-7_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1907-0

  • Online ISBN: 978-1-0716-1908-7

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