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Multicolor microRNA FISH effectively differentiates tumor types.

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.

SUBMITTER: Renwick N

PROVIDER: S-EPMC3668843 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Multicolor microRNA FISH effectively differentiates tumor types.

Renwick Neil N Cekan Pavol P Masry Paul A PA McGeary Sean E SE Miller Jason B JB Hafner Markus M Li Zhen Z Mihailovic Aleksandra A Morozov Pavel P Brown Miguel M Gogakos Tasos T Mobin Mehrpouya B MB Snorrason Einar L EL Feilotter Harriet E HE Zhang Xiao X Perlis Clifford S CS Wu Hong H Suárez-Fariñas Mayte M Feng Huichen H Shuda Masahiro M Moore Patrick S PS Tron Victor A VA Chang Yuan Y Tuschl Thomas T

The Journal of clinical investigation 20130601 6

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 overlappin ...[more]

PMID: 23728175

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Multicolor microRNA FISH effectively differentiates tumor types.

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Multicolor microRNA FISH effectively differentiates tumor types.

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