Chapter 4
Simultaneous Detection of mRNA and Protein
in S. cerevisiae by Single-Molecule FISH
and Immunofluorescence
Evelina Tutucci and Robert H. Singer
Abstract
Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of
endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA
probes hybridized against the mRNA of interest, which can be detected by using standard wide-field
fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene
expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to
correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with
immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify
two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae.
This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e.,
two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the
considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other
model organisms.
Key words smFISH, Immunofluorescence, smFISH-IF, Single molecule, RNA FISH, RNA localization, Single-cell imaging, Cell cycle, S. cerevisiae
1 Introduction
Within single cells, molecules fluctuate in a stochastic fashion
depending on their rate of synthesis and degradation. These fluctuations impact on the single-cell physiology, growth, fitness, and
phenotypic heterogeneity [1]. Part of this variability is generated
during the process of gene expression. Thus, tools yielding quantitative information on this process are of key importance to examine
the biological consequences of molecular fluctuations. To this end
the Singer lab developed single-molecule RNA fluorescent in situ
hybridization to visualize and count absolute mRNA numbers in
fixed cells [2]. This noninvasive approach allows a snapshot of intact
cells where mRNAs can be localized and quantified without losing
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020
51
Simultaneous Detection of mRNA and Protein
in S. cerevisiae by Single-Molecule FISH
and Immunofluorescence
Evelina Tutucci and Robert H. Singer
Abstract
Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of
endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA
probes hybridized against the mRNA of interest, which can be detected by using standard wide-field
fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene
expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to
correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with
immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify
two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae.
This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e.,
two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the
considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other
model organisms.
Key words smFISH, Immunofluorescence, smFISH-IF, Single molecule, RNA FISH, RNA localization, Single-cell imaging, Cell cycle, S. cerevisiae
1 Introduction
Within single cells, molecules fluctuate in a stochastic fashion
depending on their rate of synthesis and degradation. These fluctuations impact on the single-cell physiology, growth, fitness, and
phenotypic heterogeneity [1]. Part of this variability is generated
during the process of gene expression. Thus, tools yielding quantitative information on this process are of key importance to examine
the biological consequences of molecular fluctuations. To this end
the Singer lab developed single-molecule RNA fluorescent in situ
hybridization to visualize and count absolute mRNA numbers in
fixed cells [2]. This noninvasive approach allows a snapshot of intact
cells where mRNAs can be localized and quantified without losing
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020
51