Three-dimensional analysis of single molecule FISH in human colon organoids
Three-dimensional analysis of single molecule FISH in human colon organoids
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DOI：
10.1242/bio.042812
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发表时间：
2019-08-01
期刊：
影响因子：
2.4
通讯作者：
Maurice, Madelon M.
中科院分区：
文献类型：
--
作者：
Omerzu, Manja;Fenderico, Nicola;Maurice, Madelon M.
三维(3D)微型器官(类器官)的培养为研究健康和疾病状态下人体器官发育和组织自组织的基本原理提供了一个简单而强大的工具。单分子分析的应用对于全面了解组织和器官生理学的复杂性具有很高的信息量。为了充分开发单分子技术的潜力，需要对3D组织培养的方案和工具进行调整。单分子RNA荧光原位杂交(SmFISH)是一种显示和定量单个转录本的可靠技术。此外，smFISH还可用于同时研究剪接变异体、融合转录本以及多个基因的转录本。在这里，我们开发了一个为期3天的方案和验证方法，以在整个人体器官中进行3D SMFISH。我们提供了一些应用来举例说明同时检测不同的mRNA转录本、评估它们的空间分布以及在3D中识别有机物中不同的细胞谱系的各种可能性。
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.