Fully Automated Fluorescence-Based Four-Color Multiplex Assay for Co-detection of MicroRNA and Protein Biomarkers in Clinical Tissue Specimens

  • Lorenzo F. SempereEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


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.

Key words

MicroRNA (miRNA, miR) In situ hybridization (ISH) Immunohistochemistry (IHC) Biomarker Locked nucleic acid (LNA) Formalin-fixed paraffin-embedded tissue (FFPE) Breast cancer Molecular pathology Diagnostics 



I would like to thank Dr. Wendy Wells for providing financial support, technical advice, and laboratory space at the Dartmouth Pathology Translation Research Laboratory, a CAP-accredited CLIA-certified facility, in which multiplex ISH/IHC assays were conducted, and Dr. Elena Bryleva for critical reading of this manuscript. This work was supported by National Institutes of Health (NIH) and National Cancer Institute (NCI) grants R21 CA141017 and R03 CA141564, and intramural Hitchcock Foundation pilot grant.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Program in Skeletal Disease and Tumor Microenvironment, Laboratory of microRNA Diagnostics and Therapeutics, Center for Cancer and Cell BiologyVan Andel InstituteGrand RapidsUSA

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