Document resource
Background Genetic changes in mammalian genes including exon skipping and point mutations play a crucial role in the development of cancer 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 is often overexpressed and has emerged as a biomarker in various cancer types. Additionally, isoform-specific therapeutics are crucial for cancer cell survival and proliferation. Similarly, point mutations in genes such as BRAF, KRAS, p53 result in altered protein function, which can lead to cancer. Spatial visualization of gene isoform and point mutations holds the potential of providing better understanding of tumor microenvironment.Methods We developed a next-generation, fully automated fluorescent assay on the Leica platform that enables specific detection of highly similar RNA targets - such as point mutations and isoform-specific exon-exon junctions simultaneously. 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 probe on HeLa cell pellets and signal was compared to original Basescope 1zz probes. To visualize MET exon 14 skipping (METD14), MET WT exon and exon 14-15 junction specific probes were detected on the WT and variant positive cell pellets. Similarly, BRAFV600E gene point mutation was co-detected with the BRAF WT gene by using fluorescent 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 METD14 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, BRAFV600E point mutation and WT probes were duplexed in the assay and visualized using two distinct fluorophores on mutant and normal matched colon tissue. 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.