RNA Detection

Chapter 9

Detection and Automated Analysis of Single Transcripts

at Subcellular Resolution in Zebrafish Embryos

L. Carine Stapel, Coleman Broaddus, and Nadine L. Vastenhouw

Abstract

Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA
molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different
cellular compartments resulting in quantitative information on transcript levels at subcellular resolution.
The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis
pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish
cryosections. The protocol includes a method to obtain high-quality sections of zebrafish embryos, an
smFISH protocol optimized for zebrafish cryosections, and a user-friendly, automated analysis pipeline for
cell segmentation and transcript detection. The software is freely available and can be used to analyze
sections of any multicellular organism.

Key wordssmFISH, Zebrafish, Cryosections, Automated cell segmentation, Transcript detection

1 Introduction

An important feature of multicellular organisms is their large variety

of cell types. Each cell type is characterized by a specific gene

expression profile which provides information about the function

of the cell. Spatial information on gene expression is often obtained

by in situ hybridization [1, 2]. However, this technique provides

limited cellular resolution and is not quantitative due to nonlinear

signal amplification [1, 2]. Quantitative information on gene

expression is often obtained by qPCR or RNA-sequencing but

these techniques only detect highly abundant transcripts when

single cells are analyzed, and precise spatial information is lost

[3–5].

Single molecule fluorescence in situ hybridization (smFISH) is

a method to visualize single transcripts at subcellular resolution in

their original tissue context [6–11]. Stellaris smFISH is a popular

method because it is straightforward and affordable, and its quan-

titative nature has been thoroughly tested [7]. The method makes

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_9,©Springer Science+Business Media LLC 2018

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