Three-dimensional analysis of single molecule FISH in human colon organoids

Manja Omerzu; Nicola Fenderico; Buys De Barbanson; Joep Sprangers; Jeroen De Ridder; Madelon M. Maurice

doi:10.1242/bio.042812

Three-dimensional analysis of single molecule FISH in human colon organoids

Manja Omerzu, Nicola Fenderico, Buys De Barbanson, Joep Sprangers, Jeroen De Ridder, Madelon M. Maurice^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.

Original language	English
Article number	bio042812
Journal	Biology Open
Volume	8
Issue number	8
DOIs	https://doi.org/10.1242/bio.042812
Publication status	Published - 9 Aug 2019

Keywords

Differentiation
Human organoids
Smfish
Stem cells
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title = "Three-dimensional analysis of single molecule FISH in human colon organoids",

abstract = "The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.",

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author = "Manja Omerzu and Nicola Fenderico and {De Barbanson}, Buys and Joep Sprangers and {De Ridder}, Jeroen and Maurice, {Madelon M.}",

note = "Funding Information: This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. This work was supported by the European Research Council [(ERC) starting grant 242958 to M.M.M.], the European Union [Framework Programme, FP7 People: Marie-Curie Actions 608180 {\textquoteleft}WntsApp{\textquoteright} to M.M.M.] and the Netherlands Organization for Scientific Research [(NWO) VICI grant 91815604 and TOP Grant 91218050 to M.M.M.]. Funding Information: The Netherlands Organization for Scientific Research [(NWO) VICI grant 91815604 Publisher Copyright: {\textcopyright} 2019. Published by The Company of Biologists Ltd.",

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AU - De Ridder, Jeroen

AU - Maurice, Madelon M.

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N2 - The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.

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Three-dimensional analysis of single molecule FISH in human colon organoids

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