the information coming from a single cell, as it happens with global
mRNA measurements (i.e., qPCR, northern blot, RNA sequencing). By hybridizing tens of DNA probes conjugated to one or
multiple fluorophores to the mRNA of interest, enough fluorescence is concentrated to detect an mRNA as a diffraction-limited
spot. This method is not only useful to detect single mRNAs in the
cytoplasm, but it can also be used to infer the number of nascent
RNAs at a transcription site based on its fluorescence intensity
[3, 4]. Recent advancements in probe design, fluorophores, detectors, and imaging analysis improved the robustness of this technique, which is now the method of choice to precisely quantify
low-abundance mRNAs in all organisms, cell types, as well as tissues
[5–11]. Many smFISH studies revealed significant cell-to-cell variability in mRNA concentrations, which influences many cellular
processes, such as cell differentiation, development, and population
fitness [5, 12]. Also, the possibility to localize mRNAs with subcellular resolution (i.e., nucleus vs. cytoplasm [13], yeast bud [14],
polarized intestinal epithelial cells [15], or neuronal dendrites [16])
was important to understand how different stages of gene expression
from transcription to degradation are coordinated [12, 14, 16].
Nonetheless, to extend the use of smFISH to report on complex cellular states, it is important to be able to look at more than
one mRNA at the time or simultaneously look at mRNAs and
proteins within single cells. Multicolor smFISH can be easily implemented to visualize up to four distinct mRNA species labeled, i.e.,
with Cy5, Cy3.5, Cy3, or FITC fluorophores [17]. More recently,
several multiplexing single-mRNA imaging approaches have been
developed based on different barcoding schemes and repeated
rounds of hybridization, which allowed one to visualize potentially
up to thousands of different mRNAs in single yeast or mammalian
cells [18–20]. Although computationally demanding, this multiplexed imaging-based approach represents a powerful tool to perform in situ transcriptomics, complementing single-cell sequencing
methods, especially for low-abundance mRNAs. Extended discussion on these approaches can be found elsewhere [11, 21].
Alternatively, to characterize a cellular state, an mRNA and a
protein can be simultaneously visualized in single cells to gain
further information about their function. For instance, to correlate
the mRNA expression with the stage of the cell cycle, smFISH for a
cell cycle-regulated mRNA can be combined with immunofluorescence (IF) for a cell cycle marker. Here, we describe a protocol to
visualize two cell cycle-controlled mRNAs. The first is CLN2, a
cyclin expressed in late G1 phase when it associates with the cyclindependent kinase Cdc28p to activate its kinase activity [22]. The
second mRNA is ASH1, the best characterized yeast-localized
mRNA [23–26]. ASH1 mRNA is precisely expressed during anaphase, when it localizes to the bud tip to be locally translated and to
control the mating-type selection in the daughter cell of wild-type
52
Evelina Tutucci and Robert H. Singer
mRNA measurements (i.e., qPCR, northern blot, RNA sequencing). By hybridizing tens of DNA probes conjugated to one or
multiple fluorophores to the mRNA of interest, enough fluorescence is concentrated to detect an mRNA as a diffraction-limited
spot. This method is not only useful to detect single mRNAs in the
cytoplasm, but it can also be used to infer the number of nascent
RNAs at a transcription site based on its fluorescence intensity
[3, 4]. Recent advancements in probe design, fluorophores, detectors, and imaging analysis improved the robustness of this technique, which is now the method of choice to precisely quantify
low-abundance mRNAs in all organisms, cell types, as well as tissues
[5–11]. Many smFISH studies revealed significant cell-to-cell variability in mRNA concentrations, which influences many cellular
processes, such as cell differentiation, development, and population
fitness [5, 12]. Also, the possibility to localize mRNAs with subcellular resolution (i.e., nucleus vs. cytoplasm [13], yeast bud [14],
polarized intestinal epithelial cells [15], or neuronal dendrites [16])
was important to understand how different stages of gene expression
from transcription to degradation are coordinated [12, 14, 16].
Nonetheless, to extend the use of smFISH to report on complex cellular states, it is important to be able to look at more than
one mRNA at the time or simultaneously look at mRNAs and
proteins within single cells. Multicolor smFISH can be easily implemented to visualize up to four distinct mRNA species labeled, i.e.,
with Cy5, Cy3.5, Cy3, or FITC fluorophores [17]. More recently,
several multiplexing single-mRNA imaging approaches have been
developed based on different barcoding schemes and repeated
rounds of hybridization, which allowed one to visualize potentially
up to thousands of different mRNAs in single yeast or mammalian
cells [18–20]. Although computationally demanding, this multiplexed imaging-based approach represents a powerful tool to perform in situ transcriptomics, complementing single-cell sequencing
methods, especially for low-abundance mRNAs. Extended discussion on these approaches can be found elsewhere [11, 21].
Alternatively, to characterize a cellular state, an mRNA and a
protein can be simultaneously visualized in single cells to gain
further information about their function. For instance, to correlate
the mRNA expression with the stage of the cell cycle, smFISH for a
cell cycle-regulated mRNA can be combined with immunofluorescence (IF) for a cell cycle marker. Here, we describe a protocol to
visualize two cell cycle-controlled mRNAs. The first is CLN2, a
cyclin expressed in late G1 phase when it associates with the cyclindependent kinase Cdc28p to activate its kinase activity [22]. The
second mRNA is ASH1, the best characterized yeast-localized
mRNA [23–26]. ASH1 mRNA is precisely expressed during anaphase, when it localizes to the bud tip to be locally translated and to
control the mating-type selection in the daughter cell of wild-type
52
Evelina Tutucci and Robert H. Singer