Abstract
Characterizing the expression of novel targets in normal and diseased tissues is a fundamental component of a target validation data package. Often these targets are presented to the pathology team for assessment with bulk or single-cell RNAseq data and limited to no spatial tissue expression data. In situ hybridization to detect mRNA (RNAscope) is a valuable tool to (1) identify cells that may express the target protein and to corroborate protein expression during immunohistochemical (IHC) assay development or (2) to use as surrogate for single-cell expression IHC when antibodies are not available. Chromogenic RNAscope in situ hybridization (CISH) can be performed on frozen or formalin-fixed, paraffin-embedded (FFPE) tissues. This CISH workflow starts with RNA qualification of the tissue (to assess RNA integrity) by measuring the expression of housekeeping genes. RNA-qualified tissues then undergo CISH for the target in question, and positive CISH signals are quantified in VisioPharm by a combination of color deconvolution, size gating, and dot density thresholding. This RNA workflow can complement IHC or standalone in target validation for spatial characterization of novel targets.
Published Version
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