Mirvana mirna separation kit

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).

Total RNA containing small RNA was extracted from lung tissues in the control group, model group, and HC group (n=3 mice) using Trizol reagent (Invitrogen) and purified using a mirVana miRNA separation kit (Ambion, Austin, TX, USA). A spectrophotometer (NanoDrop ND-1000) was used to measure RNA purity and concentration at OD260/280. The integrity of RNA was determined by 1% formaldehyde denaturing gel electrophoresis. We used CapitalBio Technology Mouse LncRNA Array v1 (4×180K format) and the Agilent Mouse miRNA Microarray, Release 21.0 (8×60K format). Microarray experiments, including labeling and hybridization, were conducted according to the manufacturer’s instructions.