Chapter 2
Quantitative Fluorescence In Situ Hybridization Detection
of Plant mRNAs with Single-Molecule Resolution
Kun Huang, Mona Batish, Chong Teng, Alex Harkess,
Blake C. Meyers, and Jeffrey L. Caplan
Abstract
Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with
high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant
tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also
provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA
oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH
signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH
spots that reflect the copy number of target RNA.
Key words FISH, Single-molecule, In situ hybridization, Quantification, RNAs, Plant, Autofluorescence, Confocal, Linear spectral unmixing, Spectra
1 Introduction
The method of in situ hybridization is a powerful tool to add
molecular context and localization at a tissue and cellular level to
DNA [1], RNA [2], and chromosomes [3], using microscopic tools
[4]. First-generation methods for in situ hybridization in plants
detect messenger RNA (mRNA) used in vitro-transcribed digoxigenin (DIG)-labeled RNA probes. The enzymatic activity of DIG
hydrolyzes its substrates and precipitates a purple/red color at its
localization [5]. This result can be detected using bright-field light
microscopes. Fluorescent in situ hybridization (FISH) in plants was
achieved next, either by introduction of the fluorescent DIG substrates or by amplification of the DIG-labeled probes with
fluorophore-coupled antibodies. The fluorescence generated
using this method could be detected using a fluorescence widefield microscope or a laser scanning confocal microscope. However,
none of these methods provided single-molecule resolution, which
is important to study the changes in RNA transcriptional levels
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020
23
Quantitative Fluorescence In Situ Hybridization Detection
of Plant mRNAs with Single-Molecule Resolution
Kun Huang, Mona Batish, Chong Teng, Alex Harkess,
Blake C. Meyers, and Jeffrey L. Caplan
Abstract
Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with
high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant
tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also
provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA
oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH
signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH
spots that reflect the copy number of target RNA.
Key words FISH, Single-molecule, In situ hybridization, Quantification, RNAs, Plant, Autofluorescence, Confocal, Linear spectral unmixing, Spectra
1 Introduction
The method of in situ hybridization is a powerful tool to add
molecular context and localization at a tissue and cellular level to
DNA [1], RNA [2], and chromosomes [3], using microscopic tools
[4]. First-generation methods for in situ hybridization in plants
detect messenger RNA (mRNA) used in vitro-transcribed digoxigenin (DIG)-labeled RNA probes. The enzymatic activity of DIG
hydrolyzes its substrates and precipitates a purple/red color at its
localization [5]. This result can be detected using bright-field light
microscopes. Fluorescent in situ hybridization (FISH) in plants was
achieved next, either by introduction of the fluorescent DIG substrates or by amplification of the DIG-labeled probes with
fluorophore-coupled antibodies. The fluorescence generated
using this method could be detected using a fluorescence widefield microscope or a laser scanning confocal microscope. However,
none of these methods provided single-molecule resolution, which
is important to study the changes in RNA transcriptional levels
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020
23