Multicolor miRNA fluorescence in situ hybridization for tumor differential diagnosis

miRNAs are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time (RT)-PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA fluorescence in situ hybridization (FISH). For proof-of-concept, we differentiated two skin tumors, namely Basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified tumor-specific miRNAs (miR-205 and miR-375) in BCC and MCC respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and rRNA retention using model compounds and HPLC analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using pre-defined cut-off values; all were correctly identified in blinded analysis. We established a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues 6 MCC samples 2 normal skin samples were profiled with Agilent miRNA array platform