Quantitative Imaging of lncRNA Expression

Procedures were carried out as previously reported [35 (link),36 (link)] and in accordance with Stellaris fluorescence in situ hybridization (FISH) instructions (www.biocat.com (accessed on 20 July 2022)). Through an online probe design tool (www.biosearchtech.com/stellarisdesigner/ (accessed on 10 August 2022), specific probes were chosen from input sequences (PARTICLE NR_038942.1; GAS5 NR_152521.1) for optimal binding properties to the target RNA. Probe fluorophores 5′carboxyfluorescein FAM (excitation (Ex): 495 nm; emission (Em): 520 nm) were chosen for the detection of these lncRNAs. Confocal fluorescence microscopic imaging was acquired with a Leica TCS SP8X confocal system (Leica Microsystems, Mannheim, Germany) using an HCX PL APO 40_/1.30 Oil objective and appropriate excitation using 488 nm and emission using 509 nm lasers. Emitted fluorescence signals were sampled at a resolution of 30 nm/pixel with a dwell time of 1.5 μs. Image analysis was carried out as indicated above.