Cdna reverse transcription kit

Total RNA was extracted from mouse lung tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). In short, TRIzol was added to a dry powder ground on lung tissue and left to stand at room temperature for 5 min. Then, 200 μL of chloroform was added to each sample, followed by a severe upside-down vortex. After standing for 5 min, the samples were centrifuged at 12,000 rpm and 4 °C for 15 min. The samples were divided into 3 layers. The aqueous phase was reserved with 400 μL of isopropyl alcohol, left for 10 min, and centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was removed, washed with 70% ethanol, centrifuged, further dried, and then suspended in DEPC water. Finally, the concentration and purity of the extracted RNA were determined by ultraviolet spectrophotometry (One Drop, New York, NY, USA). According to the instructions of the cDNA Reverse Transcription Kit (Beyotime, Shanghai, China), primers were used to reverse-transcribe total RNA. Quantitative PCR was performed using SYBR Green (Beyotime, Shanghai, China) in a Roche LightCycler^® 480 real-time PCR system. After the reaction was completed, the period threshold (Ct) was determined, and the relative RNA levels of each sample were calculated using the 2-ΔΔCt method and normalized to the GAPDH level. The gene-specific primers used in this study are listed in Table 1.