mRNA accumulation and protein production [4], mRNA measurements provide information about the rate of transcription, accumulation, and decay, revealing modes of gene expression regulation.
Bulk mRNA measurements, i.e., northern blots, quantitative PCR,
and RNA sequencing, are informative to measure multiple mRNA
species from a single RNA preparation and to perform relative
comparisons of mRNA levels in different conditions. However,
these approaches, which average millions of cells, have fundamental
limitations when it comes to precisely measuring RNAs at the level
of single cells, or within cellular compartments, i.e.,
nucleus vs. cytoplasm, cellular protrusions such as the bud of
S. cerevisiae, the leading edge of fibroblast, or neuronal dendrites
and axons.
To achieve quantitative subcellular mRNA measurements, several approaches based on fluorescence microscopy have been developed over the past decades. In fixed cells, a standard method to
visualize and count individual mRNAs is single-molecule fluorescent in situ hybridization (smFISH) [5, 6]. Briefly, this approach
allows detecting single endogenous mRNAs by hybridizing tens of
fluorescently labeled DNA oligos onto the target molecule. By
using sensitive digital cameras and wide-field microscopy, it is possible to detect single mRNAs as diffraction-limited spots, allowing
their subcellular localization and quantification in thousands of
cells. This method can be applied to single isolated cells (i.e., [7–
13]) as well as to tissues [14–16]. Several modified smFISH protocols exist, which use DNA probes of different lengths and complexities (i.e., 20 mer, 50 mer, branched DNA, RNA scope [8, 10,
17]) or fluorescence amplification systems to detect weak signals
(i.e., hybridization chain reaction, HCR [18]). An important difference between these techniques is whether the probes are fluorescently labeled or not, in which case they need to be detected with a
secondary fluorescent oligo. A simple, reliable, and affordable protocol that uses indirect labeling has recently been published
[19]. Furthermore, smFISH can be multiplexed to simultaneously
visualize different mRNA species within single cells (up to 10,000;
[15, 20–24]), or it can also be combined to protein detection by
immunofluorescence (see refs. 25, 26 and this issue). These
approaches revealed asymmetric RNA distribution within single
cells, as well as significant cell-to-cell variability existing in tissue
or even isogenic populations [27–32]. Furthermore, by using
smFISH, other aspects of gene expression have been characterized,
such as the “bursty” nature of transcription [8, 9, 33, 34], the
mechanisms controlling mRNA export form the nucleus to the
cytoplasm [35, 36], as well as the control of mRNA degradation
[37, 38]. For more in-depth reviews see refs. 1, 2, 39; see also
Chapter 1 by Bleckmann et al. and Chapter 4 by Tutucci and
Singer.
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Xavier Pichon et al.
Bulk mRNA measurements, i.e., northern blots, quantitative PCR,
and RNA sequencing, are informative to measure multiple mRNA
species from a single RNA preparation and to perform relative
comparisons of mRNA levels in different conditions. However,
these approaches, which average millions of cells, have fundamental
limitations when it comes to precisely measuring RNAs at the level
of single cells, or within cellular compartments, i.e.,
nucleus vs. cytoplasm, cellular protrusions such as the bud of
S. cerevisiae, the leading edge of fibroblast, or neuronal dendrites
and axons.
To achieve quantitative subcellular mRNA measurements, several approaches based on fluorescence microscopy have been developed over the past decades. In fixed cells, a standard method to
visualize and count individual mRNAs is single-molecule fluorescent in situ hybridization (smFISH) [5, 6]. Briefly, this approach
allows detecting single endogenous mRNAs by hybridizing tens of
fluorescently labeled DNA oligos onto the target molecule. By
using sensitive digital cameras and wide-field microscopy, it is possible to detect single mRNAs as diffraction-limited spots, allowing
their subcellular localization and quantification in thousands of
cells. This method can be applied to single isolated cells (i.e., [7–
13]) as well as to tissues [14–16]. Several modified smFISH protocols exist, which use DNA probes of different lengths and complexities (i.e., 20 mer, 50 mer, branched DNA, RNA scope [8, 10,
17]) or fluorescence amplification systems to detect weak signals
(i.e., hybridization chain reaction, HCR [18]). An important difference between these techniques is whether the probes are fluorescently labeled or not, in which case they need to be detected with a
secondary fluorescent oligo. A simple, reliable, and affordable protocol that uses indirect labeling has recently been published
[19]. Furthermore, smFISH can be multiplexed to simultaneously
visualize different mRNA species within single cells (up to 10,000;
[15, 20–24]), or it can also be combined to protein detection by
immunofluorescence (see refs. 25, 26 and this issue). These
approaches revealed asymmetric RNA distribution within single
cells, as well as significant cell-to-cell variability existing in tissue
or even isogenic populations [27–32]. Furthermore, by using
smFISH, other aspects of gene expression have been characterized,
such as the “bursty” nature of transcription [8, 9, 33, 34], the
mechanisms controlling mRNA export form the nucleus to the
cytoplasm [35, 36], as well as the control of mRNA degradation
[37, 38]. For more in-depth reviews see refs. 1, 2, 39; see also
Chapter 1 by Bleckmann et al. and Chapter 4 by Tutucci and
Singer.
122
Xavier Pichon et al.