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Quantitative Fluorescence In Situ Hybridization Detection of Plant mRNAs with Single-Molecule Resolution

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RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH spots that reflect the copy number of target RNA.

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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

    Article  CAS  Google Scholar 

  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

    Article  Google Scholar 

  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

    Article  CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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-070317-033037

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  Google Scholar 

  9. Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol 714:3–13. https://doi.org/10.1007/978-1-61779-005-8_1

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rosa S, Duncan S, Dean C (2016) Mutually exclusive sense-antisense transcription at FLC facilitates environmentally induced gene repression. Nat Commun 7:13031

    Article  CAS  Google Scholar 

  12. Huang K, Baldrich P, Meyers BC, Caplan JL (2019) sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants. Plant J 98:359–369. https://doi.org/10.1111/tpj.14210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/Nmeth.2019

    Article  CAS  Google Scholar 

  14. Mueller F, Senecal A, Tantale K, Marie-Nelly H, Ly N, Collin O, Basyuk E, Bertrand E, Darzacq X, Zimmer C (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4):277–278

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations

  1. Department of Plant and Soil Sciences, University of Delaware, Newark, DE, USA

    Kun Huang & Jeffrey L. Caplan

  2. Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA

    Kun Huang & Jeffrey L. Caplan

  3. Departments of Biological Sciences and Medical & Molecular Sciences, University of Delaware, Newark, DE, USA

    Mona Batish

  4. Donald Danforth Plant Science Center, St. Louis, MO, USA

    Chong Teng, Alex Harkess & Blake C. Meyers

Authors
  1. Kun Huang
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  2. Mona Batish
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  3. Chong Teng
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  4. Alex Harkess
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  5. Blake C. Meyers
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  6. Jeffrey L. Caplan
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Corresponding author

Correspondence to Jeffrey L. Caplan .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Huang, K., Batish, M., Teng, C., Harkess, A., Meyers, B.C., Caplan, J.L. (2020). Quantitative Fluorescence In Situ Hybridization Detection of Plant mRNAs with Single-Molecule Resolution. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

  • eBook Packages: Springer Protocols

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