ribosomal protein 17mRNA (MusRpl17) as a negative control
for maize tissue.
6. The concentration of the probe that should be applied varies
from sample to sample. The starting concentration should be
1 ng/μL. Increase the concentration if the obtained smFISH
signal is too weak. If high background is observed, decrease the
probe concentration first to 0.3 ng/μL, and then to 0.1 ng/μL.
7. For confocal microscopes that do not have an emission online
fingerprinting mode, a spectral image could be acquired and
then linear unmixed using the positive and autofluorescence
spectra.
Acknowledgments
This project was supported by the US NSF Plant Genome Research
Program, awards 1649424, 1611853, and 1754097. We would like
to thank members of the Batish lab for input on single-molecule in
situ hybridization, and members of the Meyers and Caplan labs for
help and support. Microscopy equipment was acquired with a
shared instrumentation grant (S10 OD016361) and access was
supported by the NIH-NIGMS (P20 GM103446), the NSF
(IIA-1301765), and the State of Delaware.
References
1. Anamthawatjonsson K, Reader SM (1995)
Pre-annealing of total genomic DNA probes
for simultaneous genomic in-situ hybridization. Genome 38(4):814–816
2. Maluszynska J, Schweizer D (1989) Ribosomal
RNA genes in B chromosomes of Crepis capillaris detected by non-radioactive in situ hybridization. Heredity 62(Pt 1):59–65
3. Fransz PF, Stam M, Montijn B, TenHoopen R,
Wiegant J, Kooter JM, Oud O, Nanninga N
(1996) Detection of single-copy genes and
chromosome rearrangements in Petunia
hybrida by fluorescence in situ hybridization.
Plant J 9(5):767–774
4. Weiss H, Pasierbek P, Maluszynska J (2000) An
improved nonfluorescent detection system for
in situ hybridization in plants. Biotech Histochem 75(2):49–53
5. Trinh le A, McCutchen MD, Bonner-Fraser M,
Fraser SE, Bumm LA, McCauley DW (2007)
Fluorescent in situ hybridization employing
the conventional NBT/BCIP chromogenic
stain. BioTechniques 42(6):756–759. https://
doi.org/10.2144/000112476
6. Taniguchi Y, Choi PJ, Li GW, Chen H,
Babu M, Hearn J, Emili A, Xie XS (2010)
Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single
cells. Science 329(5991):533–538. https://
doi.org/10.1126/science.1188308
7. 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
8. 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
9. Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol
714:3–13. https://doi.org/10.1007/978-161779-005-8_1
10. Vargas DY, Shah K, Batish M, Levandoski M,
Sinha S, Marras SA, Schedl P, Tyagi S (2011)
Single-molecule imaging of transcriptionally
coupled and uncoupled splicing. Cell 147
(5):1054–1065. https://doi.org/10.1016/j.
cell.2011.10.024
11. Rosa S, Duncan S, Dean C (2016) Mutually
exclusive sense-antisense transcription at FLC
32
Kun Huang et al.
for maize tissue.
6. The concentration of the probe that should be applied varies
from sample to sample. The starting concentration should be
1 ng/μL. Increase the concentration if the obtained smFISH
signal is too weak. If high background is observed, decrease the
probe concentration first to 0.3 ng/μL, and then to 0.1 ng/μL.
7. For confocal microscopes that do not have an emission online
fingerprinting mode, a spectral image could be acquired and
then linear unmixed using the positive and autofluorescence
spectra.
Acknowledgments
This project was supported by the US NSF Plant Genome Research
Program, awards 1649424, 1611853, and 1754097. We would like
to thank members of the Batish lab for input on single-molecule in
situ hybridization, and members of the Meyers and Caplan labs for
help and support. Microscopy equipment was acquired with a
shared instrumentation grant (S10 OD016361) and access was
supported by the NIH-NIGMS (P20 GM103446), the NSF
(IIA-1301765), and the State of Delaware.
References
1. Anamthawatjonsson K, Reader SM (1995)
Pre-annealing of total genomic DNA probes
for simultaneous genomic in-situ hybridization. Genome 38(4):814–816
2. Maluszynska J, Schweizer D (1989) Ribosomal
RNA genes in B chromosomes of Crepis capillaris detected by non-radioactive in situ hybridization. Heredity 62(Pt 1):59–65
3. Fransz PF, Stam M, Montijn B, TenHoopen R,
Wiegant J, Kooter JM, Oud O, Nanninga N
(1996) Detection of single-copy genes and
chromosome rearrangements in Petunia
hybrida by fluorescence in situ hybridization.
Plant J 9(5):767–774
4. Weiss H, Pasierbek P, Maluszynska J (2000) An
improved nonfluorescent detection system for
in situ hybridization in plants. Biotech Histochem 75(2):49–53
5. Trinh le A, McCutchen MD, Bonner-Fraser M,
Fraser SE, Bumm LA, McCauley DW (2007)
Fluorescent in situ hybridization employing
the conventional NBT/BCIP chromogenic
stain. BioTechniques 42(6):756–759. https://
doi.org/10.2144/000112476
6. Taniguchi Y, Choi PJ, Li GW, Chen H,
Babu M, Hearn J, Emili A, Xie XS (2010)
Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single
cells. Science 329(5991):533–538. https://
doi.org/10.1126/science.1188308
7. 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
8. 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
9. Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol
714:3–13. https://doi.org/10.1007/978-161779-005-8_1
10. Vargas DY, Shah K, Batish M, Levandoski M,
Sinha S, Marras SA, Schedl P, Tyagi S (2011)
Single-molecule imaging of transcriptionally
coupled and uncoupled splicing. Cell 147
(5):1054–1065. https://doi.org/10.1016/j.
cell.2011.10.024
11. Rosa S, Duncan S, Dean C (2016) Mutually
exclusive sense-antisense transcription at FLC
32
Kun Huang et al.