Quantitative RT-PCR Analysis of Gene Expression

Frozen lung tissue was ground to a fine powder using a mortar and pestle. RNA from isolated alveolar macrophages or lung tissue were purified using the RNeasy mini kit as per manufacturers instruction (Qiagen, GMH Germany). The NanoDrop200 (Thermo Fischer Scientific, United States) was used to determine the concentration and purity of extracted RNA and the High-Capacity RNA to cDNA kit (Thermo Fischer Scientific, United States) was used to generate complementary DNA (cDNA). RT-qPCR was performed using the QuantStudio 7 system (Thermo Fischer Scientific, United States), TaqMan primers and the Taqman Fast Advanced Master Mix, as per manufacturer’s instructions. The relative expression of each gene was normalised against the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) and determined using the ΔΔCt value method, as previously described (Wang et al., 2018 (link); Wang et al., 2019 (link)).