Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jun 15, 2018
- Source ID
- 10.1242/dev.165753
Entities
People
- Alexandre Cunha
- Aneesh Acharya
- Georgios Artavanis
- Harry Choi
- Johannes Stegmaier
- Maayan Schwarzkopf
- Mark E. Fornace
- Niles A. Pierce
Organizations
- Balliol College
- California Institute of Technology
- Defense Advanced Research Projects Agency
- German Research Foundation
- Gordon and Betty Moore Foundation
- Karlsruhe Institute of Technology
- National Institute of Biomedical Imaging and Bioengineering
- National Institutes of Health
- National Science Foundation
- RWTH Aachen University
- University of Oxford