Lighting up microRNAs
In recent years, microRNAs (miRNAs) and other non-coding RNAs have emerged as disease biomarkers. miRNA profiles have been used to establish tissue origin for cancers of unknown primary origin, determine prognosis, monitor therapeutic responses and screen for disease, but clinically tractable, diagnostic methods for monitoring miRNA expression in patient samples are not currently available. Neil Renwick, Pavol Cekan, and colleagues developed a multicolor miRNA fluorescence in situ hybridization (FISH) technology that can be used to visualize miRNAs in formalin-fixed paraffin-embedded (FFPE) tissue sections, such as those collected from patients. Using this method, Renwick and colleagues were able to identify tumor specific miRNAs in basal cell carcinoma and Merkel cell carcinoma (accompanying image) and distinguish between FFPE sections from the two tumor types. This proof-of concept study indicates that fluorescent RNA FISH could serve as a molecular diagnostic in a clinical setting.
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Abstract
MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, 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 the 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 ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (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 predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.
Authors
Neil Renwick, Pavol Cekan, Paul A. Masry, Sean E. McGeary, Jason B. Miller, Markus Hafner, Zhen Li, Aleksandra Mihailovic, Pavel Morozov, Miguel Brown, Tasos Gogakos, Mehrpouya B. Mobin, Einar L. Snorrason, Harriet E. Feilotter, Xiao Zhang, Clifford S. Perlis, Hong Wu, Mayte Suárez-Fariñas, Huichen Feng, Masahiro Shuda, Patrick S. Moore, Victor A. Tron, Yuan Chang, Thomas Tuschl
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ISSN: 0021-9738 (print), 1558-8238 (online)