Locked nucleic acid-in situ hybridization (LNA-ISH) with tyramide signal amplification (TSA) was performed to detect lncRNA and miRNA as previously described13 (link),69 (link). All LNA probes were synthesized (Exiqon) including double biotin-labeled probe against MIR100HG, double digoxigenin (DIG)-labeled probe against miR-125b, double fluorescein-labeled probe against miR-100, DIG-labeled probe against U6 snRNA, and DIG-labeled scramble probe. Anti-Digoxigenin HRP Conjugate, anti-Fluorescein HRP Conjugate, Streptavidin-HRP Conjugate, and TSA Cy3 and Fluorescein Kit (all from PerkinElmer) were used for TSA methods. Confocal fluorescence microscopy was performed using a Zeiss LSM 710 confocal microscope.
For detection of MET amplification, MET/CEP7 dual-color probes (Cytotest) were used for recognizing the MET gene status following the manufacturer's protocol. Analysis was according to the University of Colorado Cancer Center (UCCC) criteria. A MET/CEP7 ratio was established based on counting at least 200 cells.
For detection of MET amplification, MET/CEP7 dual-color probes (Cytotest) were used for recognizing the MET gene status following the manufacturer's protocol. Analysis was according to the University of Colorado Cancer Center (UCCC) criteria. A MET/CEP7 ratio was established based on counting at least 200 cells.