RNA was extracted as reported in our previous study27. Briefly, the lungs from HDM- or normal-saline-treated mice were incubated with Sepasol-RNA I Super G solution for RNA isolation (Nacalai Tesque, Osaka, Japan) and homogenized with metal beads in a multi-bead shocker (Yasui Kikai, Osaka, Japan). Then, the crushed samples were added to 200 μL of chloroform and centrifuged at 15,300×g at 4 °C for 15 min. The supernatant was collected, and 500 μL of isopropanol was added. The sample was then mixed well, and the mixture was centrifuged at 15,300×g at 4 °C for 10 min. The supernatant was discarded, and 70% ethanol was added to the nucleic acid pellets. The pellet was then centrifuged at 15,300×g at 4 °C for 5 min. The supernatant was discarded, and the nucleic acid pellet was dried and dissolved in 200 μL of water.

cDNA was synthesized from the extracted RNA using ReverTra Ace qPCR Master Mix (Toyobo, Osaka, Japan).

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