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Single-Molecule RNA FISH in Whole-Mount Organoids


Borrelli, Costanza; Moor, Andreas E (2020). Single-Molecule RNA FISH in Whole-Mount Organoids. In: Ordóñez-Morán, Paloma. Intestinal Stem Cells. Nottingham, UK: Springer, 237-247.

Abstract

Single-molecule RNA fluorescent in situ hybridization (smFISH) enables the detection and quantification of single RNA molecules. Three-dimensional organoid cultures have emerged as versatile in vitro primary culture models that recapitulate many physiological features of their tissue of origin. Here we describe a protocol to visualize single RNA molecules in organoid cultures. Our method accommodates both a whole-mount staining workflow which requires spinning disk confocal microscopy, and a cryosectioning workflow which is compatible with widefield microscopy. Organoid smFISH enables to address various biological problems that range from the identification of cell types (e.g., via the intestinal stem cell marker Lgr5) to the quantification of RNA localization in an epithelium.

Abstract

Single-molecule RNA fluorescent in situ hybridization (smFISH) enables the detection and quantification of single RNA molecules. Three-dimensional organoid cultures have emerged as versatile in vitro primary culture models that recapitulate many physiological features of their tissue of origin. Here we describe a protocol to visualize single RNA molecules in organoid cultures. Our method accommodates both a whole-mount staining workflow which requires spinning disk confocal microscopy, and a cryosectioning workflow which is compatible with widefield microscopy. Organoid smFISH enables to address various biological problems that range from the identification of cell types (e.g., via the intestinal stem cell marker Lgr5) to the quantification of RNA localization in an epithelium.

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Additional indexing

Contributors:Intestinal Stem Cells
Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:2020
Deposited On:09 Feb 2021 13:44
Last Modified:10 Feb 2021 21:00
Publisher:Springer
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-0716-0747-3_15
PubMed ID:32705646

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