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Updated: Jan 21, 2026

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
Published on: September 20, 2020
Three-dimensional analysis of single molecule FISH in human colon organoids
Manja Omerzu1, Nicola Fenderico1, Buys de Barbanson2,3
1Oncode Institute and Department of Cell Biology, Centre for Molecular Medicine, University Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands.
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 distinct mRNA transcripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.
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