title: 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.
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Renwick
authors.forename: Pavol
authors.initials: P
authors.lastname: Cekan
authors.forename: Paul A
authors.initials: PA
authors.lastname: Masry
authors.forename: Sean E
authors.initials: SE
authors.lastname: McGeary
authors.forename: Jason B
authors.initials: JB
authors.lastname: Miller
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
authors.forename: Zhen
authors.initials: Z
authors.lastname: Li
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Mihailovic
authors.forename: Pavel
authors.initials: P
authors.lastname: Morozov
authors.forename: Miguel
authors.initials: M
authors.lastname: Brown
authors.forename: Tasos
authors.initials: T
authors.lastname: Gogakos
authors.forename: Mehrpouya B
authors.initials: MB
authors.lastname: Mobin
authors.forename: Einar L
authors.initials: EL
authors.lastname: Snorrason
authors.forename: Harriet E
authors.initials: HE
authors.lastname: Feilotter
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.forename: Clifford S
authors.initials: CS
authors.lastname: Perlis
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Mayte
authors.initials: M
authors.lastname: Suárez-Fariñas
authors.forename: Huichen
authors.initials: H
authors.lastname: Feng
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shuda
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Moore
authors.forename: Victor A
authors.initials: VA
authors.lastname: Tron
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chang
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002280:Basal Cell Carcinoma / Q000175:diagnosis* / Q000378:metabolism; D015266:Carcinoma, Merkel Cell / Q000175:diagnosis* / Q000378:metabolism; D016000:Cluster Analysis; D003937:Diagnosis, Differential; D005404:Fixatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D025202:Molecular Diagnostic Techniques; D016612:Paraffin Embedding; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059629:Signal-To-Noise Ratio; D012878:Skin Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005404:Fixatives; D005456:Fluorescent Dyes; D005557:Formaldehyde; D035683:MicroRNAs; D012339:RNA, Ribosomal, 28S; C534425:MIR205, human; C540933:MIRN375 microRNA, human