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In Situ Hybridization Protocols pp 143–156Cite as

Multiplexed Quantitative In Situ Hybridization for Mammalian Cells on a Slide: qHCR and dHCR Imaging (v3.0)

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2148))

Abstract

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in diverse sample types. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution. Here, we provide protocols for qHCR and dHCR imaging in mammalian cells on a slide.

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References

  1. Choi HMT, Schwarzkopf M, Fornace ME et al (2018) Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145:dev165753. https://doi.org/10.1242/dev.165753

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Acknowledgments

We thank M.E. Fornace for developing the Dot Analysis 1.0 software package for analyzing dHCR images. Within the Beckman Institute at Caltech, we thank the following for assistance: C.R. Calvert and G.J. Shin (Molecular Technologies), A. Collazo and S. Wilbert (Biological Imaging Facility), and J. Stegmaier and A. Cunha (Center for Advanced Methods in Image Analysis). This work was funded by the National Institutes of Health (National Institute of Biomedical Imaging and Bioengineering R01EB006192), by the Defense Advanced Research Projects Agency (HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government), by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC), by the Gordon and Betty Moore Foundation (GBMF2809), by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by a Professorial Fellowship at Balliol College, University of Oxford, and by the Eastman Visiting Professorship at the University of Oxford.

Competing Interests

The authors declare competing financial interests in the form of patents, pending patent applications, and a startup company (Molecular Instruments).

Author information

Authors and Affiliations

  1. Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Maayan Schwarzkopf, Harry M. T. Choi & Niles A. Pierce

  2. Division of Engineering & Applied Science, California Institute of Technology, Pasadena, CA, USA

    Niles A. Pierce

  3. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Niles A. Pierce

Authors
  1. Maayan Schwarzkopf
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  2. Harry M. T. Choi
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  3. Niles A. Pierce
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Corresponding author

Correspondence to Niles A. Pierce .

Editor information

Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

  2. Cancer Research UK, University of Cambridge, Cambridge, UK

    Julia Jones

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Schwarzkopf, M., Choi, H.M.T., Pierce, N.A. (2020). Multiplexed Quantitative In Situ Hybridization for Mammalian Cells on a Slide: qHCR and dHCR Imaging (v3.0). In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0623-0_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0622-3

  • Online ISBN: 978-1-0716-0623-0

  • eBook Packages: Springer Protocols

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