miRNA in situ hybridization in circulating tumor cells - MishCTC

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.JJDM thanks the Spanish Ministerio de Economía y Competitividad for a Ramón y Cajal Fellowship (Grant CTQ2012-34778). This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Program (FP7-PEOPLE-2011-CIG-Project Number 294142 to RMSM and FP7-PEOPLE-2012-CIG-Project Number 322276 toJJDM). Thisresearch wasalso partially supported by Consejeria de Salud de la Junta de Andalucı ́ a (PI0294-2012)

Selective protein and DNA adsorption on PLL-PEG films modulated by ionic strength.

We describe a soft thin film which selectively adsorbs DNA but averts the non-specific binding of proteins. Indium tin oxide (ITO) substrates were surface-modified with a poly(L-lysine)-g-poly(ethylene glycol) (PLL-PEG) film which carries an outer protein-repelling PEG layer and an underlying positively charged PLL layer that attracts DNA. Binding of DNA could be tuned by a factor of over 90 by varying the salt concentration. The dependence of DNA binding on ionic strength was described with a physicochemical model which led to the conclusion of an unexpectedly high enrichment of salt inside the PEG layer. In addition, the model led to an expanded definition of the Debye-Huckel type effective screening length parameter z. Our new findings on a film with dual passivation/attraction properties can find applications in biopolymer-specific coatings useful in bioseparation and biosensing. In addition, the physicochemical characterisation provides new insight into the interactions between biopolymers and polymer-coated interfaces