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Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0)

Choi, Harry M. T. and Schwarzkopf, Maayan and Pierce, Niles A. (2020) Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0). In: In Situ Hybridization Protocols. Methods in Molecular Biology. No.2148. Humana Press , New York, NY, pp. 159-178. ISBN 978-1-0716-0622-3. https://resolver.caltech.edu/CaltechAUTHORS:20200518-142723478

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Abstract

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in the anatomical context of whole-mount vertebrate embryos. 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 in an anatomical context and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution in an anatomical context. Here, we provide protocols for qHCR and dHCR imaging in whole-mount zebrafish, chicken, and mouse embryos.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
https://doi.org/10.1007/978-1-0716-0623-0_10DOIArticle
ORCID:
AuthorORCID
Choi, Harry M. T.0000-0002-1530-0773
Pierce, Niles A.0000-0003-2367-4406
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 and V. Trivedi for developing the Read-out/Read-in 1.0 software package for performing read-out/read-in analyses on multiplexed qHCR images in vertebrate embryos. 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).
Funders:
Funding AgencyGrant Number
NIHR01EB006192
Defense Advanced Research Projects Agency (DARPA)HR0011-17-2-0008
Caltech Beckman InstituteUNSPECIFIED
Gordon and Betty Moore FoundationGBMF2809
NSFCCF-1317694
University of OxfordUNSPECIFIED
Subject Keywords:Accuracy; Autofluorescence; Automatic background suppression; dHCR imaging; Fluorescence in situ hybridization (FISH); Hybridization chain reaction (HCR); In situ HCR v3.0; mRNA imaging; Multiplexed; Precision; qHCR imaging; Quantitative; Signal amplification; Single-molecule imaging; Thick sample penetration; Whole-mount vertebrate embryos
Series Name:Methods in Molecular Biology
Issue or Number:2148
Record Number:CaltechAUTHORS:20200518-142723478
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200518-142723478
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103286
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:18 May 2020 21:37
Last Modified:18 May 2020 21:37

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