miRNA in situ hybridization in circulating tumor cells - MishCTC Ortega, Francisco G. Lorente Acosta, José Antonio García Puche, José Luis Ruiz, María P. Sánchez Martín, Rosario María Miguel-Pérez, Diego de Díaz Mochón, Juan José Serrano Fernández, María José Fluorescence imaging Metastasis irculating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype. 2015-04-17T06:30:42Z 2015-04-17T06:30:42Z 2015 info:eu-repo/semantics/article Ortega, F.G.; et al. miRNA in situ hybridization in circulating tumor cells - MishCTC. Scientific Reports, 5: 9207 (2015). [http://hdl.handle.net/10481/35704] 2045-2322 http://hdl.handle.net/10481/35704 10.1038/srep09207 eng info:eu-repo/grantAgreement/EC/FP7/294142 info:eu-repo/grantAgreement/EC/FP7/322276 http://creativecommons.org/licenses/by-nc-nd/3.0/ info:eu-repo/semantics/openAccess Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Nature Publishing Group