Fully automated fluorescence-based four-color multiplex assay for co-detection of microRNA and protein biomarkers in clinical tissue specimens.

The application of locked nucleic acid chemistry for microRNA detection by in situ hybridization, and thereby visualization of microRNA expression at single-cell resolution, has contributed to our understanding of the roles that these short noncoding regulatory RNAs play during development, physiology, and disease. Several groups have implemented chromogenic-based and fluorescence-based protocols to detect microRNA expression in formalin-fixed paraffin-embedded clinical tissue specimens. These emerging robust and reproducible tissue slide-based assays are valid tools to bring about the clinical application of in situ microRNA detection for routine diagnostics. Here, I describe a fully automated fluorescence-based four-color multiplex assay for co-detection of a microRNA (e.g., let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210), reference RNA (e.g., U6 snRNA, 18S rRNA), and protein markers (e.g., CD11b, CD20, CD45, collagen I, cytokeratin 7, cytokeratin 19, smooth muscle actin, tubulin, vimentin) in FDA-approved Leica Bond-MAX staining station.