# Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging

> **NIH NIH R44** · MOLECULAR INSTRUMENTS, INC. · 2021 · $150,000

## Abstract

Project Summary
Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging
Encoded in the genome of each organism, biological circuits direct development, maintain integrity in the face
of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. RNA in
situ hybridization (RNA-ISH) and immunohistochemistry (IHC) methods provide biologists, drug developers, and
pathologists with critical windows into the spatial organization of this circuitry, enabling imaging of mRNA and
protein expression in an anatomical context. While it is desirable to perform multiplexed experiments in which
a panel of targets is imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH
and IHC methods in highly autofluorescent samples including whole-mount vertebrate embryos and FFPE tissue
sections, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of
reporter molecules, and staining is non-quantitative. These multi-decade technological shortcomings are signifi-
cant impediments to biological research as well as to advancement in drug development and pathology assays,
preventing high-dimensional quantitative analyses of developmental and disease-relevant regulatory networks in
an anatomical context.
 To overcome these challenges, in situ amplification based on the mechanism of hybridization chain reaction
(HCR) draws on concepts from the emerging discipline of dynamic nucleic acid nanotechnology to achieve four
RNA-ISH breakthroughs in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain
slices, and FFPE tissue sections: 1) straightforward multiplexing with 1-step quantitative signal amplification
for up to 5 target mRNAs simultaneously; 2) analog mRNA relative quantitation with subcellular resolution in
an anatomical context; 3) digital mRNA absolute quantitation with single-molecule resolution in an anatomical
context; 4) automatic background suppression throughout the protocol, dramatically enhancing performance and
ease-of-use. The proposed research will build on the unique capabilities of HCR to enable next-generation
levels of multiplexing for RNA-ISH, to extend the benefits of 1-step multiplexed quantitative enzyme-free HCR
signal amplification to IHC, and to develop the first in situ amplification product for simultaneous RNA-ISH/IHC,
performing 1-step HCR signal amplification for targets RNAs and proteins simultaneously. During Phase I, we
will engineer molecular components to enable highly multiplexed signal amplification, and establish a unified
framework for RNA and protein imaging. During Phase II, we will develop and commercialize robust technologies
for highly multiplexed RNA-ISH/IHC in key sample types critical for academic research, drug development, and/or
clinical diagnostics. These new HCR products will enable biologists, drug developers, and pathologists to perform
previously impossible ...

## Key facts

- **NIH application ID:** 10158198
- **Project number:** 1R44GM140796-01
- **Recipient organization:** MOLECULAR INSTRUMENTS, INC.
- **Principal Investigator:** Harry Ming Tak Choi
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $150,000
- **Award type:** 1
- **Project period:** 2021-04-01 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10158198

## Citation

> US National Institutes of Health, RePORTER application 10158198, Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging (1R44GM140796-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10158198. Licensed CC0.

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