Chapter 1
Tagging and Application of RNA Probes
for Sequence-Specific Visualization of RNAs by Fluorescent
In Situ Hybridization
Thomas Dresselhaus and Andrea Bleckmann
Abstract
To understand the development and differentiation processes within a tissue and a cell, analysis of the cell
type-specific gene expression pattern as well as the subcellular localization of the produced RNAs is
essential. The simplest and fastest method to visualize RNA molecules is in situ hybridization (ISH) on
whole-tissue samples. Over the past 40 years, various labeling and visualization techniques have been
established to analyze either the expression domain of genes in tissues (using the classical chromogenic
detection system) or the specific subcellular localization of mRNAs (using fluorescently labeled probes). By
using the Arabidopsis root tip as an example tissue, we describe and compare classic in situ hybridization
techniques. The protocols described can be easily transferred to almost all other tissues or model organism
with slight modifications.
Key words Fluorescent RNA in situ hybridization, Whole mount, FISH, TSA, Subcellular RNA
detection, Root, Arabidopsis
1 Introduction
RNA in situ hybridization is the most accurate method to study
gene expression patterns and is superior to approaches such as RNA
detection by Northern blots lacking cellular resolution or
promoter-marker studies that are often misleading due to stability
differences of makers, their products, and the RNA of interest.
RNA in situ hybridization is especially applied to study spatial and
temporal gene expression patterns during development, but also in
differentiated tissues [1, 2]. Classical RNA in situ hybridization is a
rather cheap and fast method to analyze specific gene expression in
tissues or at the subcellular level in a 2–3-day experiment. In this
chapter, we describe and compare three different detection systems
for whole-mount RNA in situ hybridization (ISH) to visualize
specific RNAs in the root tip of Arabidopsis thaliana (Fig. 1). In
an ISH experiment the cells are fixed, and the in vivo-produced
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020
3
Tagging and Application of RNA Probes
for Sequence-Specific Visualization of RNAs by Fluorescent
In Situ Hybridization
Thomas Dresselhaus and Andrea Bleckmann
Abstract
To understand the development and differentiation processes within a tissue and a cell, analysis of the cell
type-specific gene expression pattern as well as the subcellular localization of the produced RNAs is
essential. The simplest and fastest method to visualize RNA molecules is in situ hybridization (ISH) on
whole-tissue samples. Over the past 40 years, various labeling and visualization techniques have been
established to analyze either the expression domain of genes in tissues (using the classical chromogenic
detection system) or the specific subcellular localization of mRNAs (using fluorescently labeled probes). By
using the Arabidopsis root tip as an example tissue, we describe and compare classic in situ hybridization
techniques. The protocols described can be easily transferred to almost all other tissues or model organism
with slight modifications.
Key words Fluorescent RNA in situ hybridization, Whole mount, FISH, TSA, Subcellular RNA
detection, Root, Arabidopsis
1 Introduction
RNA in situ hybridization is the most accurate method to study
gene expression patterns and is superior to approaches such as RNA
detection by Northern blots lacking cellular resolution or
promoter-marker studies that are often misleading due to stability
differences of makers, their products, and the RNA of interest.
RNA in situ hybridization is especially applied to study spatial and
temporal gene expression patterns during development, but also in
differentiated tissues [1, 2]. Classical RNA in situ hybridization is a
rather cheap and fast method to analyze specific gene expression in
tissues or at the subcellular level in a 2–3-day experiment. In this
chapter, we describe and compare three different detection systems
for whole-mount RNA in situ hybridization (ISH) to visualize
specific RNAs in the root tip of Arabidopsis thaliana (Fig. 1). In
an ISH experiment the cells are fixed, and the in vivo-produced
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020
3