Multiomic Detection of RNA, Protein, and Protein-Protein Proximity at Subcellular Resolution Using Automated Protease-Free RNAscope™ Workflows.

Regulation of gene expression occurs at multiple levels from RNA synthesis to protein turnover. Understanding the complexity requires integrating information from genome, epigenome, transcriptome, proteome, and metabolome layers. Although no single technology currently allows simultaneous analysis of all molecular modalities, in situ methods that uniquely offer both single-cell resolution and spatial context have been adapted to generate multiomic readouts Here, we describe an advanced multiomic in situ hybridization (ISH) approach that integrates RNA, protein, and protein-proximity readouts-capturing spatial relationships between protein targets-on the same tissue section. We introduce a novel protease-free workflow utilizing RNAscope chemistry and oligo conjugated antibody (OCAs), enabling detection of protease-sensitive protein epitopes without compromising RNA detection sensitivity. This integrated method facilitates comprehensive, spatially resolved multiomic analysis crucial for investigating cell neighborhoods and interactions in complex biological tissues.