CaltechAUTHORS
Published May 12, 2020 | Version public
Book Section - Chapter Metadata-only

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

Creators

  • 1. ROR icon California Institute of Technology
  • 2. ROR icon MRC Weatherall Institute of Molecular Medicine

Contributors

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.

Additional Information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature. First Online: 12 May 2020. 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).

Additional details

Identifiers

Eprint ID
103287
Resolver ID
CaltechAUTHORS:20200518-143331174

Funding

NIH
R01EB006192
Defense Advanced Research Projects Agency (DARPA)
HR0011-17-2-0008
Caltech Beckman Institute
Gordon and Betty Moore Foundation
GBMF2809
NSF
CCF-1317694
University of Oxford

Dates

Created
2020-05-18
Created from EPrint's datestamp field
Updated
2021-11-16
Created from EPrint's last_modified field

Caltech Custom Metadata

Caltech groups
Division of Biology and Biological Engineering (BBE)
Series Name
Methods in Molecular Biology
Series Volume or Issue Number
2148