Mirna microarray chip

Microarray expression profiling was performed using 5 µg total RNA extracted from peripheral blood mononuclear cells (PBMCs) collected from 22 cases and 19 controls. MiRCURYTM Array Labeling kit (Exiqon, Vedbaek, Denmark) was utilized to label miRNAs fluorescently, and then miRNA microarray chips (Exiqon) were utilized to hybridize miRNAs. Hybridization data were collected using GenePix 4000B laser scanner (Axon Instruments, Foster City, CA, USA), and GenePix 4.0 software (Axon Instruments) was utilized to digitize and analyze the images.

Cells were lysed in TRIzol reagent (#15596026, Thermo Fisher Scientific) to isolate total RNA. miRNA from the total RNA sample was isolated using a mirVana miRNA separation kit (AM1561, Ambion, Austin, TX, USA). The quality and concentration of the extracted miRNA were examined using a spectrophotometer (Bio-Rad Inc.) and 1% formaldehyde-agarose gel electrophoresis. miRNA expression was determined using miRNA microarray chips (Exiqon, Vedbaek, Denmark). In short, total RNA (5 μg) was subjected to Cy3 modification at the RNA 3ʹend using the T4 RNA ligase (EL0021, Thermo Fisher Scientific). This reaction was performed at 37°C for 30 min. After that, the labeled RNA was hybridized with Human miRNA Expression Microarray V4.0 (Arraystar, Rockville, MD, USA) for 24 h. The gene expression data were obtained using a GeneChip^TM Scanner 3000 7 G system (#00-0210, 2008, Thermo Fisher Scientific) and analyzed by the R Language Program (Version 3.6.3, R).