# Background free amplified single-molecule FISH for in situ and flow cytometric applications

> **NIH NIH R01** · RBHS-NEW JERSEY MEDICAL SCHOOL · 2020 · $557,328

## Abstract

Abstract
 Cellular RNAs can serve as powerful biomarkers of diverse disease states. Recent developments
in probe technology enable the detection of RNAs with single-molecule sensitivity by fluorescence in situ
hybridization (sm-FISH). This technology holds great promise in clinical diagnostics for the detection of
pathogens and cancers. However, its utility for imaging pathological sections is presently limited, because
in order to obtain single-molecule sensitivity, imaging needs to be performed with high magnification objectives
that have a limited field of view, permitting the observation of only a few cells at a time. The relatively low
intensity of these signals also limits the utility of sm-FISH when the cells are analyzed by flow cytometry,
because rare cells, such as immunological memory cells or stem cells, which express a low level of mRNA
markers, cannot be detected. A further limitation is that single nucleotide variations cannot be detected.
 We are developing a new generation of sm-FISH in which the signals is amplified without any
amplification of background. In our approach, that we refer to as strictly target-dependent amplified FISH
(stamp-FISH), when a pair of binary probes bind to the target a sequestered sequence is exposed. The
exposed sequence then serves as an initiator of a hybridization chain reaction, which leads to creation of a
large, highly fluorescent DNA cluster that remains tethered at the target. We obtain as much as 10-fold
amplification of signal without any enhancement in background. Furthermore, the probes yield exquisite
discrimination between single nucleotide variations.
 We will develop this technology further and apply it for detection of point mutations in EGFR and BRAF
mRNAs. Currently, about fifteen percent of the copies of target mRNA that are present within the cells can be
detected. We will improve this efficiency using strategies aimed at increasing the binding of probes to the
target mRNAs. Probe sets will be developed for an exemplary set of mutations within the EGFR and BRAF
genes and then cell lines and cancer tissues, in which these mutations are expressed, will be imaged using
these probe sets. We will also develop multiplex assays that will detect three mutants and the wild-type mRNA
simultaneously. In addition, for T cell profiling we are proposing to detect 15 RNA markers in multiplex in
T cells. With this expansion of the multiplexing range to 15 targets, we will be able to detect subtypes of cells
that differ from each other by the expression of chemokine/cytokines, transcription factors, or metabolic
markers. This development will create powerful new analytical possibilities for diverse fields in both research
and clinical settings and thus will be of broad utility.

## Key facts

- **NIH application ID:** 9844937
- **Project number:** 5R01CA227291-03
- **Recipient organization:** RBHS-NEW JERSEY MEDICAL SCHOOL
- **Principal Investigator:** SANJAY TYAGI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $557,328
- **Award type:** 5
- **Project period:** 2018-02-16 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9844937

## Citation

> US National Institutes of Health, RePORTER application 9844937, Background free amplified single-molecule FISH for in situ and flow cytometric applications (5R01CA227291-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9844937. Licensed CC0.

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