17. While acquiring the Z-stacks for the different channels, start
imaging from the longest (CY5) to the shortest (DAPI) wavelengths and finish with the DIC. Long exposure times can
cause photo-bleaching. To increase the signal of the smFISH,
it is better to increase the number of probes specific for each
mRNA rather than increasing the exposure time for signal
imaging for longer than 1 s.
18. Three representative datasets and smFISH analysis files are
available for download and are described in [33, 34].
Acknowledgments
We would like to thank Carolina Eliscovich and Noura Ghazale for
helpful discussion during the protocol development. This work was
supported by NIH Grant GM57071 to R.H.S. E.T. was supported
by
Swiss
National
Science
Foundation
Fellowships
P2GEP3_155692 and P300PA_164717.
Contributions: E.T. designed the protocol, performed the experiments, and analyzed the data. E.T. and R.H.S. wrote the
manuscript.
References
1. Bruggeman FJ, Teusink B (2018) Living with
noise: On the propagation of noise from molecules to phenotype and fitness. Curr Opin Syst
Biol 8:144–150
2. Femino AM, Fay FS, Fogarty K, Singer RH
(1998) Visualization of single RNA transcripts
in situ. Science 280(5363):585–590
3. Zenklusen D, Larson DR, Singer RH (2008)
Single-RNA counting reveals alternative modes
of gene expression in yeast. Nat Struct Mol Biol
15(12):1263–1271.
https://doi.org/10.
1038/nsmb.1514
4. Raj A, Peskin CS, Tranchina D, Vargas DY,
Tyagi S (2006) Stochastic mRNA synthesis in
mammalian cells. PLoS Biol 4(10):e309.
https://doi.org/10.1371/journal.pbio.
0040309
5. Tutucci E, Livingston NM, Singer RH, Wu B
(2018) Imaging mRNA in vivo, from birth to
death. Annu Rev Biophys 47:85–106. https://
doi.org/10.1146/annurev-biophys-070317033037
6. Pichon X, Bastide A, Safieddine A, Chouaib R,
Samacoits A, Basyuk E, Peter M, Mueller F,
Bertrand E (2016) Visualization of single
endogenous polysomes reveals the dynamics
of translation in live human cells. J Cell Biol
214(6):769–781. https://doi.org/10.1083/
jcb.201605024
7. Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging
technology. Nat Methods 8(4 Suppl):
S12–S19. https://doi.org/10.1038/nmeth.
1573
8. Farack L, Egozi A, Itzkovitz S (2018) Single
molecule approaches for studying gene regulation in metabolic tissues. Diabetes Obes Metab
20(Suppl 2):145–156. https://doi.org/10.
1111/dom.13390
9. Raj A, van den Bogaard P, Rifkin SA, van
Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly
labeled probes. Nat Methods 5(10):877–879.
https://doi.org/10.1038/nmeth.1253
10. Long X, Colonell J, Wong AM, Singer RH,
Lionnet T (2017) Quantitative mRNA imaging throughout the entire Drosophila brain.
Nat Methods 14(7):703–706. https://doi.
org/10.1038/nmeth.4309
Detection of mRNA and Proteins in Single Cells by smFISH-IF
67
imaging from the longest (CY5) to the shortest (DAPI) wavelengths and finish with the DIC. Long exposure times can
cause photo-bleaching. To increase the signal of the smFISH,
it is better to increase the number of probes specific for each
mRNA rather than increasing the exposure time for signal
imaging for longer than 1 s.
18. Three representative datasets and smFISH analysis files are
available for download and are described in [33, 34].
Acknowledgments
We would like to thank Carolina Eliscovich and Noura Ghazale for
helpful discussion during the protocol development. This work was
supported by NIH Grant GM57071 to R.H.S. E.T. was supported
by
Swiss
National
Science
Foundation
Fellowships
P2GEP3_155692 and P300PA_164717.
Contributions: E.T. designed the protocol, performed the experiments, and analyzed the data. E.T. and R.H.S. wrote the
manuscript.
References
1. Bruggeman FJ, Teusink B (2018) Living with
noise: On the propagation of noise from molecules to phenotype and fitness. Curr Opin Syst
Biol 8:144–150
2. Femino AM, Fay FS, Fogarty K, Singer RH
(1998) Visualization of single RNA transcripts
in situ. Science 280(5363):585–590
3. Zenklusen D, Larson DR, Singer RH (2008)
Single-RNA counting reveals alternative modes
of gene expression in yeast. Nat Struct Mol Biol
15(12):1263–1271.
https://doi.org/10.
1038/nsmb.1514
4. Raj A, Peskin CS, Tranchina D, Vargas DY,
Tyagi S (2006) Stochastic mRNA synthesis in
mammalian cells. PLoS Biol 4(10):e309.
https://doi.org/10.1371/journal.pbio.
0040309
5. Tutucci E, Livingston NM, Singer RH, Wu B
(2018) Imaging mRNA in vivo, from birth to
death. Annu Rev Biophys 47:85–106. https://
doi.org/10.1146/annurev-biophys-070317033037
6. Pichon X, Bastide A, Safieddine A, Chouaib R,
Samacoits A, Basyuk E, Peter M, Mueller F,
Bertrand E (2016) Visualization of single
endogenous polysomes reveals the dynamics
of translation in live human cells. J Cell Biol
214(6):769–781. https://doi.org/10.1083/
jcb.201605024
7. Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging
technology. Nat Methods 8(4 Suppl):
S12–S19. https://doi.org/10.1038/nmeth.
1573
8. Farack L, Egozi A, Itzkovitz S (2018) Single
molecule approaches for studying gene regulation in metabolic tissues. Diabetes Obes Metab
20(Suppl 2):145–156. https://doi.org/10.
1111/dom.13390
9. Raj A, van den Bogaard P, Rifkin SA, van
Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly
labeled probes. Nat Methods 5(10):877–879.
https://doi.org/10.1038/nmeth.1253
10. Long X, Colonell J, Wong AM, Singer RH,
Lionnet T (2017) Quantitative mRNA imaging throughout the entire Drosophila brain.
Nat Methods 14(7):703–706. https://doi.
org/10.1038/nmeth.4309
Detection of mRNA and Proteins in Single Cells by smFISH-IF
67