Related Experiment Video

Updated: Jun 1, 2025

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay
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Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Published on: August 14, 2018

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Integration of Chromogenic RNAscope In Situ Hybridization for Target Validation in Drug Discovery

Rosanna Win1, Wesley Minto1, In Kyoung Mah1

  • 1Gilead Sciences, Foster City, California, USA.

Toxicologic Pathology
|January 20, 2025
PubMed

Insights

Novel RNAscope in situ hybridization (ISH) methods enable spatial characterization of gene expression in tissues. This technique complements immunohistochemistry (IHC) for target validation and spatial analysis.

Area of Science:

  • Biopathology
  • Molecular Pathology
  • Genomics

Background:

  • Target validation requires characterizing novel targets in normal and diseased tissues.
  • Traditional methods like RNA sequencing lack spatial expression data.
  • Immunohistochemistry (IHC) is common but requires specific antibodies.

Purpose of the Study:

  • To present a Chromogenic RNAscope in situ hybridization (CISH) workflow for spatial gene expression analysis.
  • To demonstrate CISH as a valuable tool for target validation and spatial characterization.
  • To highlight CISH as a complement or alternative to IHC.

Main Methods:

  • RNAscope ISH workflow on frozen or FFPE tissues.
  • Initial RNA quality assessment using housekeeping genes.
  • CISH for target mRNA detection.
  • Quantification of CISH signals using VisioPharm software (color deconvolution, size gating, dot density thresholding).

Main Results:

  • The CISH workflow successfully detects and quantifies mRNA expression spatially within tissues.
  • RNA qualification ensures reliable assay performance.
  • Quantification methods allow for objective assessment of target expression.

Conclusions:

  • CISH is an effective method for spatial characterization of novel targets.
  • This workflow can support IHC assay development or serve as a surrogate for single-cell IHC.
  • The presented RNA workflow is valuable for target validation in pathology.
Keywords:
RNAscopeimmunohistochemistryin situ hybridizationquantitative image analysistarget validation

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