Basescope™ duplex assay enables same-slide detection of short RNA targets including isoforms

Background Genetic changes in mammalian genes including exon skipping and point mutations play a crucial role in oncogenesis and predicting patient survival and disease prognosis. Studies have shown that cancer cell lines can be distinguished from non-cancer cell line based on gene transcript isoform. As example, MET (receptor tyrosine kinase) exon 14 skipping mutation and EGFR exon 19, 21 deletion or exon 20 insertion mutation have emerged as a biomarker in various cancer types. Additionally, alternative-splicing plays a crucial role in disease progression and therapeutic response. Spatial visualization of gene isoform holds the potential of providing better understanding of tumor microenvironment. Methods We developed a next-generation, manual and fully automated fluorescent assays on the Leica platform that enables specific and simultaneous detection of highly similar RNA targets - isoform-specific exon-exon junctions. The assay utilizes specificity and sensitivity of RNAscope TM technology to visualize single RNA molecule on formalin fixed paraffin embedded (FFPE) tissue and cell pellets. To validate the new workflow, endogenous control genes were detected using 1zz probes on HeLa cell pellets and Mouse multi tissue array. Signal and background were quantitatively compared to original BaseScope 1zz probes. To visualize MET exon 14 skipping (METΔ14), MET WT exon and exon 14-15 junction specific probes were detected on the WT and variant positive cell pellets. Further, exon 13-15 or exon 14-15 junction probe and exon 15 probes were duplexed and co-detected in MET WT and Δ14 cell pellets, respectively using BaseScope duplex assay that utilizes streamlined RNAscope chemistry to potentially co-detect mRNAs, proteins and protein-protein interactions. Results Signal (as determined by dot counts/cell) from control gene using new BaseScope technology was comparable to signal generated using original BaseScope assay. Next, we visualized specific signal from METΔ14 exon junction probe on MET mutant pellet and WT MET probe on WT cell pellet, using dual fluorophores to identify both isoforms on the same slide. No signal was observed from mutant probe in WT pellet indicating specificity of the assay. Similarly, MET exon 14-15 junction probe and exon 15 probes were duplexed in the assay and visualized using two distinct fluorophores on wild-type cell pellet. Exon 13-15 junction probe and exon 15 probe were duplexed and co-detected using two fluorophores on mutant cell pellet. These results demonstrate the assay's efficacy in same-slide detection of cells expressing two short targets. Conclusions This BaseScope Duplex assay provides spatial resolution of isoform and point mutation-specific gene expression, offering a powerful tool for exploring cell-specific transcript variants within the tumor microenvironment and potentially advancing precision oncology research.