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Published June 26, 2018 | Submitted + Published + Supplemental Material
Journal Article Open

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


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.

Additional Information

© 2018 Published by The Company of Biologists Ltd. Received 21 March 2018; Accepted 2 May 2018. Published 26 June 2018. We thank C. R. Calvert for assistance with test tube validation of split-initiator probes, C. L. Beisel for the gift of HEK d2eGFP cells, A. Z. Rosenthal for the gift of E. coli K12 MG1655 cells and E. coli K12 MG1655 pUA66-sdhC cells expressing gfpmut2, J. Tan-Cabugao and M. Simons Costa in the M. Bronner Lab for preparation of chicken embryos, C.R. Calvert and G.J. Shin of Molecular Technologies for synthesis of HCR amplifiers, A. Collazo and S. Wilbert of the Caltech Biological Imaging Facility (BIF) for assistance with imaging, R. Diamond and D. Perez of the Caltech Flow Cytometry Facility (FCF) for assistance with flow cytometry, and M. Mann and the D. Baltimore Lab for generously providing access to their flow cytometer for preliminary studies. Competing interests: The authors declare competing financial interests in the form of patents, pending patent applications and a startup company (Molecular Instruments). Author contributions: Conceptualization: H.M.T.C., N.A.P.; Methodology: H.M.T.C., M.S., M.E.F., N.A.P.; Software: M.E.F., J.S., A.C.; Validation: H.M.T.C., M.S.; Investigation: H.M.T.C., M.S., A.A., G.A.; Writing - original draft: H.M.T.C., M.S., N.A.P.; Writing - review & editing: H.M.T.C., M.S., M.E.F., A.A., G.A., J.S., A.C., N.P.; Visualization: H.M.T.C., M.S., N.A.P.; Supervision: N.A.P.; Project administration: N.A.P.; Funding acquisition: N.A.P. This work was funded by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC; Center for Advanced Methods in Biological Image Analysis, CAMBIA), by Defense Advanced Research Projects Agency (HR0011-17-2-0008), by the Gordon and Betty Moore Foundation (GBMF2809, GBMF3406), by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by the National Institutes of Health (National Institute of Biomedical Imaging and Bioengineering R01EB006192 and National Research Service Award T32 GM007616), by the Deutsche Forschungsgemeinschaft (MI1315/4-1), by a Professorial Fellowship at Balliol College, University of Oxford, and by the Eastman Visiting Professorship at the University of Oxford. The findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government. Deposited in PMC for release after 12 months.

Attached Files

Published - dev165753.full.pdf

Submitted - 285213.full.pdf

Supplemental Material - DEV165753supp.pdf


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August 19, 2023
October 20, 2023