20. After being processed, each movie should be checked to ensure
that no drift or loss of focus happened during the acquisition
(especially for long-term movies).
Acknowledgments
This work was supported by NIH Grant GM57071 to R.H.S., and
by an ANRS Grant to E.B. E.T. was supported by Swiss National
Science Foundation Fellowships P2GEP3_155692 and
P300PA_164717. X.P. was supported by a fellowship from the
Labex EpiGenMed Montpellier/Universite ´ de Montpellier, and
E.B. had a travel grant from the Philippe Foundation.
Contributions: X.P. performed the experiments and wrote the protocol for mammalian cells. M.C.R. performed the experiments
testing different FP in yeast. E.T. performed experiments and
designed the protocol for yeast. E.T. wrote the manuscript with
inputs from E.B. and R.H.S.
References
1. 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
2. Vera M, Biswas J, Senecal A, Singer RH, Park
HY (2016) Single-cell and single-molecule
analysis of gene expression regulation. Annu
Rev Genet 50:267–291. https://doi.org/10.
1146/annurev-genet-120215-034854
3. Raj A, van Oudenaarden A (2008) Nature,
nurture, or chance: stochastic gene expression
and its consequences. Cell 135(2):216–226.
https://doi.org/10.1016/j.cell.2008.09.050
4. Liu Y, Beyer A, Aebersold R (2016) On the
dependency of cellular protein levels on mRNA
abundance. Cell 165(3):535–550. https://doi.
org/10.1016/j.cell.2016.03.014
5. Femino AM, Fay FS, Fogarty K, Singer RH
(1998) Visualization of single RNA transcripts
in situ. Science 280(5363):585–590
6. Femino AM, Fogarty K, Lifshitz LM,
Carrington W, Singer RH (2003) Visualization
of single molecules of mRNA in situ. Methods
Enzymol 361:245–304
7. Gandhi SJ, Zenklusen D, Lionnet T, Singer
RH (2011) Transcription of functionally
related constitutive genes is not coordinated.
Nat Struct Mol Biol 18(1):27–34. https://doi.
org/10.1038/nsmb.1934
8. 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
9. Senecal A, Munsky B, Proux F, Ly N, Braye FE,
Zimmer C, Mueller F, Darzacq X (2014) Transcription factors modulate c-Fos transcriptional
bursts. Cell Rep 8(1):75–83. https://doi.org/
10.1016/j.celrep.2014.05.053
10. 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
11. Yang L, Titlow J, Ennis D, Smith C, Mitchell J,
Young FL, Waddell S, Ish-Horowicz D, Davis I
(2017) Single molecule fluorescence in situ
hybridisation
for
quantitating
posttranscriptional regulation in Drosophila brains.
Methods 126:166–176. https://doi.org/10.
1016/j.ymeth.2017.06.025
12. Buxbaum AR, Wu B, Singer RH (2014) Single
beta-actin mRNA detection in neurons reveals
a mechanism for regulating its translatability.
Science 343:419–422. https://doi.org/10.
1126/science.1242939
13. Duncan S, Olsson TSG, Hartley M, Dean C,
Rosa S (2016) A method for detecting single
mRNA molecules in Arabidopsis thaliana.
Plant Methods 12:13. https://doi.org/10.
1186/s13007-016-0114-x
140
Xavier Pichon et al.
that no drift or loss of focus happened during the acquisition
(especially for long-term movies).
Acknowledgments
This work was supported by NIH Grant GM57071 to R.H.S., and
by an ANRS Grant to E.B. E.T. was supported by Swiss National
Science Foundation Fellowships P2GEP3_155692 and
P300PA_164717. X.P. was supported by a fellowship from the
Labex EpiGenMed Montpellier/Universite ´ de Montpellier, and
E.B. had a travel grant from the Philippe Foundation.
Contributions: X.P. performed the experiments and wrote the protocol for mammalian cells. M.C.R. performed the experiments
testing different FP in yeast. E.T. performed experiments and
designed the protocol for yeast. E.T. wrote the manuscript with
inputs from E.B. and R.H.S.
References
1. 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
2. Vera M, Biswas J, Senecal A, Singer RH, Park
HY (2016) Single-cell and single-molecule
analysis of gene expression regulation. Annu
Rev Genet 50:267–291. https://doi.org/10.
1146/annurev-genet-120215-034854
3. Raj A, van Oudenaarden A (2008) Nature,
nurture, or chance: stochastic gene expression
and its consequences. Cell 135(2):216–226.
https://doi.org/10.1016/j.cell.2008.09.050
4. Liu Y, Beyer A, Aebersold R (2016) On the
dependency of cellular protein levels on mRNA
abundance. Cell 165(3):535–550. https://doi.
org/10.1016/j.cell.2016.03.014
5. Femino AM, Fay FS, Fogarty K, Singer RH
(1998) Visualization of single RNA transcripts
in situ. Science 280(5363):585–590
6. Femino AM, Fogarty K, Lifshitz LM,
Carrington W, Singer RH (2003) Visualization
of single molecules of mRNA in situ. Methods
Enzymol 361:245–304
7. Gandhi SJ, Zenklusen D, Lionnet T, Singer
RH (2011) Transcription of functionally
related constitutive genes is not coordinated.
Nat Struct Mol Biol 18(1):27–34. https://doi.
org/10.1038/nsmb.1934
8. 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
9. Senecal A, Munsky B, Proux F, Ly N, Braye FE,
Zimmer C, Mueller F, Darzacq X (2014) Transcription factors modulate c-Fos transcriptional
bursts. Cell Rep 8(1):75–83. https://doi.org/
10.1016/j.celrep.2014.05.053
10. 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
11. Yang L, Titlow J, Ennis D, Smith C, Mitchell J,
Young FL, Waddell S, Ish-Horowicz D, Davis I
(2017) Single molecule fluorescence in situ
hybridisation
for
quantitating
posttranscriptional regulation in Drosophila brains.
Methods 126:166–176. https://doi.org/10.
1016/j.ymeth.2017.06.025
12. Buxbaum AR, Wu B, Singer RH (2014) Single
beta-actin mRNA detection in neurons reveals
a mechanism for regulating its translatability.
Science 343:419–422. https://doi.org/10.
1126/science.1242939
13. Duncan S, Olsson TSG, Hartley M, Dean C,
Rosa S (2016) A method for detecting single
mRNA molecules in Arabidopsis thaliana.
Plant Methods 12:13. https://doi.org/10.
1186/s13007-016-0114-x
140
Xavier Pichon et al.