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Development. 2018 Jun 26;145(12). pii: dev165753. doi: 10.1242/dev.165753.

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.

Author information

1
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
2
Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
3
Center for Advanced Methods in Biological Image Analysis, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.
4
Institute for Automation & Applied Informatics, Karlsruhe Institute of Technology, Karlsruhe 76344, Germany.
5
Institute of Imaging & Computer Vision, RWTH Aachen University, Aachen 52074, Germany.
6
Center for Data-Driven Discovery, California Institute of Technology, Pasadena, CA 91125, USA.
7
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA niles@caltech.edu.
8
Division of Engineering & Applied Science, California Institute of Technology, Pasadena, CA 91125, USA.
9
Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Abstract

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.

KEYWORDS:

Automatic background suppression; In situ HCR v3.0; Multiplexed quantitative in situ hybridization; dHCR imaging; qHCR flow cytometry; qHCR imaging

PMID:
29945988
PMCID:
PMC6031405
[Available on 2019-06-15]
DOI:
10.1242/dev.165753
[Indexed for MEDLINE]

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