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In-situ Hybridization

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...

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Related Experiment Video

Updated: Sep 9, 2025

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Published on: July 6, 2022

Dual probe ligation in situ hybridization with rolling-circle amplification for high-plex spatial transcriptomics.

Sarah E Maguire¹, Joel Credle², Elizabeth M W Bertelson¹

¹Portal Bioscience, LLC, Baltimore, MD, 21205, USA.

Biochemistry and Biophysics Reports

|September 2, 2025

Summary

This summary is machine-generated.

This study introduces Ligation In Situ Hybridization followed by rolling circle amplification (LISH-LnR), a new spatial transcriptomics method. It accurately maps mRNA in degraded formalin-fixed and paraffin-embedded tissues, advancing biological insights from clinical specimens.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Understanding tissue architecture is crucial for biological insights.
Spatial transcriptomics technologies are vital for studying tissue architecture.
Formalin-fixed and paraffin-embedded (FFPE) tissues are widely available but challenging for transcriptomics due to mRNA degradation.

Purpose of the Study:

To develop a streamlined method for spatial transcriptomics analysis of FFPE tissues.
To accurately detect specific mRNA isoforms within FFPE tissue architectures.
To enable highly multiplexed spatial transcriptomic studies on clinical specimens.

Main Methods:

Ligation In Situ Hybridization followed by rolling circle amplification (LISH-LnR) was developed.
Iterative fluorescent probe hybridization and imaging were employed for multiplexed detection.
Molecular rheostats were used to fine-tune assay performance.

Main Results:

LISH-LnR accurately detects spatial locations of specific mRNA isoforms in FFPE tissues.
The method was successfully demonstrated on FFPE specimens from inclusion body myositis and pediatric rhabdomyosarcoma patients.
The LISH-LnR assay performance was successfully fine-tuned using molecular rheostats.

Conclusions:

LISH-LnR provides a powerful toolkit for spatial transcriptomics on FFPE clinical specimens.
This methodology enhances the analysis of degraded mRNA in challenging tissue types.
Combined with LISH-seq and LISH-QC, LISH-LnR advances the field of spatial transcriptomics.