Title | Multicolor microRNA FISH effectively differentiates tumor types. |
Publication Type | Journal Article |
Year of Publication | 2013 |
Authors | Renwick N, Cekan P, Masry PA, McGeary SE, Miller JB, Hafner M, Li Z, Mihailovic A, Morozov P, Brown M, Gogakos T, Mobin MB, Snorrason EL, Feilotter HE, Zhang X, Perlis CS, Wu H, Suárez-Fariñas M, Feng H, Shuda M, Moore PS, Tron VA, Chang Y, Tuschl T |
Journal | J Clin Invest |
Volume | 123 |
Issue | 6 |
Pagination | 2694-702 |
Date Published | 2013 Jun |
ISSN | 1558-8238 |
Keywords | Animals, Biomarkers, Tumor, Carcinoma, Basal Cell, Carcinoma, Merkel Cell, Cluster Analysis, Diagnosis, Differential, Fixatives, Fluorescent Dyes, Formaldehyde, Gene Expression, Humans, In Situ Hybridization, Fluorescence, Mice, Mice, Knockout, MicroRNAs, Molecular Diagnostic Techniques, Paraffin Embedding, RNA, Ribosomal, 28S, Sequence Analysis, RNA, Signal-To-Noise Ratio, Skin Neoplasms, Tissue Fixation |
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. |
DOI | 10.1172/JCI68760 |
Alternate Journal | J. Clin. Invest. |
PubMed ID | 23728175 |
PubMed Central ID | PMC3668843 |
Grant List | R01 CA159227 / CA / NCI NIH HHS / United States K08 NS072235 / NS / NINDS NIH HHS / United States R01 CA170354 / CA / NCI NIH HHS / United States UL1RR024143 / RR / NCRR NIH HHS / United States R01 CA136363 / CA / NCI NIH HHS / United States UL1 RR024143 / RR / NCRR NIH HHS / United States R33 CA120726 / CA / NCI NIH HHS / United States / / Howard Hughes Medical Institute / United States R01 MH080442 / MH / NIMH NIH HHS / United States T32 GM007739 / GM / NIGMS NIH HHS / United States R01 CA120726 / CA / NCI NIH HHS / United States |
Submitted by kej2006 on June 6, 2018 - 4:09pm