Sorvall legend micro 17r

The samples were thawed on ice and then centrifuged for 5 minutes at 1,6000 x g at a temperature of 4°C (Sorvall Legend Micro 17R, Thermo Scientific) to remove cryoprecipitates. Soon after, 200 μL of the supernatant was transferred to a new 2-mL microtube. The total RNA extraction process was conducted using the miRNeasy Serum/Plasma kit (Qiagen, Germany) according to the manufacturer’s instructions.
To monitor the efficiency of total RNA extraction, 0.02 pmol/μl synthetic miRNA-39-3p (Caenorhabditis elegans—cel-miR-39-3p; Thermo Scientific) was added to the sample during the extraction process, as recommended by the manufacturer.
The extracted RNA was eluted with 14 μL of nuclease-free water (Qiagen, Germany). The concentration and purity of the RNA were assessed using a spectrophotometer (Nanodrop 2000, Thermo Scientific). Due to the low concentration of circulating miRNA in serum, cDNA synthesis was performed using the Taqman® Advanced miRNA Assay kit for low-throughput samples (Thermo Scientific) according to the protocol provided by the manufacturer.

BALF and serum analysis was conducted as described in our previous study [26 (link)]. The animal study was conducted twice to verify reproducibility. In the first study, three mice were used for the evaluation of BALF and serum, with the remaining four mice used for other studies, such as the morphological analysis (H&E stain and Masson's trichrome stain) and the expression of specific proteins related to the occurrence of COPD (IHC). BALF collection was scheduled from all mice after anesthetization with 50 mg/kg Zoletil (Virbac, Carros, France), and the tracheas were cannulated with disposable animal feeding needles. Lavage was performed with three 0.4 mL aliquots of cold phosphate-buffered saline (PBS), and BALF samples were collected and immediately centrifuged at 3,000 rpm for 5 min (Sorvall Legend Micro 17R, Thermo Fisher Scientific Inc., Waltham, MA, USA). The cell pellets were resuspended in PBS, and total and differential cell counts were obtained. The number of total cells and differential cells was counted with a Hemavet Multispecies Hematology System (Drew Scientific Inc., Waterbury, CT, USA). After cell collection (if used), the animals were sacrificed by an additional Zoletil injection. IgE levels in the serum were measured using a specific mouse IgE ELISA kit (BD Bioscience, catalog number 555248, San Jose, CA, USA) in accordance with the manufacturer's protocols.

For the metabolomic analysis, primary urine samples were prepared using published procedures (Liu et al., 2015 (link)). Urine samples were collected at 24 h intervals on 4°C gel ice packs (TECHNI ICE^™, Frankston, Victoria, Australia) and processed by centrifugation at 12,000 × g for 10 min at 4°C (Sorvall Legend Micro 17R, Thermo Fischer Scientific, Inc., Waltham, MA, United States). Prior to the LC-MS analysis, each lyophilized sample was dissolved in a 0.1% formic acid-acetonitrile solution (95:5), and a 5 µL aliquot was used as the injected volume.