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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Acknowledgments
We thank Gerald Schwank, Nina Frey, and Jan Reichmuth for generous donations of pancreatic and colon cancer organoid samples. We thank Efi Massasa and Shalev Itzkovitz for help with creating the organoid cryosectioning protocol. We thank Dario Zimmerli, Nikolaos Doumpas, and Matthias Moor for valuable comments on the manuscript. AEM is funded by the Swiss National Science Foundation grant PCEPP3_181249.
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Borrelli, C., Moor, A.E. (2020). Single-Molecule RNA FISH in Whole-Mount Organoids. In: Ordóñez-Morán, P. (eds) Intestinal Stem Cells. Methods in Molecular Biology, vol 2171. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0747-3_15
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DOI: https://doi.org/10.1007/978-1-0716-0747-3_15
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