Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

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Item Type:	Article
Title:	Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos
Creators Name:	Stapel, L.C., Lombardot, B., Broaddus, C., Kainmueller, D., Jug, F., Myers, E.W. and Vastenhouw, N.L.
Abstract:	Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.
Keywords:	Cell Segmentation, Gene Expression, Image Analysis Pipeline, smFISH, Animals, Zebrafish
Source:	Development
ISSN:	0950-1991
Publisher:	Company of Biologists
Volume:	143
Number:	3
Page Range:	540-546
Date:	1 February 2016
Official Publication:	https://doi.org/10.1242/dev.128918
PubMed:	View item in PubMed

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