Abstract 2094: Development of a multiomic workflow using Oncore Pro X to stain tissues for high performance Dual Channel™ Validation of RNA expression with downstream quantitation using AI-based image analysis with Oncotopix Discovery™

Abstract Accurate quantification of RNA expression remains a critical challenge in advancing RNA-ISH multiomic technologies for translational and clinical applications. Current barriers include lack of guidelines for assay validation and difficulties in correlating RNA abundance with spatial localization. In this study, we introduce a robust pipeline leveraging Dual Channel™ Validation (DCV) of HCR™ HiFi Probes and AI-enabled analysis to enable relative quantification of RNA abundance. The DCV approach uses two spectrally distinct fluorescent channels to confirm the specificity and sensitivity of RNA detection. This dual-channel strategy minimizes false positives while providing a reliable proxy for RNA transcript abundance through fluorescent intensity correlations. To enhance RNA quantitation, our pipeline integrates automated tissue staining for consistent and high-quality labeling plus advanced AI-based algorithms trained on combined cellular nuclear plus membrane segmentation markers to precisely delineate cell boundaries. This dual-marker framework addresses limitations of rule based nuclear expansion-based segmentation methods and improves the accuracy of RNA spot assignment. Our results demonstrate that DCV, combined with autostaining and AI-enabled cellular segmentation, achieves superior quantification fidelity. This approach not only validates RNA-ISH assay performance but also provides a scalable, reproducible method for relative RNA quantitation across diverse tissue types. By coupling high-performance probes with optimized automated tissue staining (on Oncore Pro X) and advanced computational analysis, our pipeline bridges critical gaps in RNA quantification, facilitating reliable, clinically meaningful insights into RNA expression within complex tissue environments. Citation Format: Randy Chen, Aneesh Acharya, Daniel Winkowski, Jason Ramos, Harry Choi, Regan Baird. Development of a multiomic workflow using Oncore Pro X to stain tissues for high performance Dual Channel™ Validation of RNA expression with downstream quantitation using AI-based image analysis with Oncotopix Discovery™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2094.