RNA Extraction and cDNA Synthesis

The method for RNA extraction was demonstrated from our previous reports^{9 (link),58 (link)}. 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 then homogenized with metal beads in a multi-bead shocker (Yasui Kikai, Osaka, Japan). The crushed samples were added to 200μL chloroform and then centrifuged at 15,300×g at 4 °C for 15 min. The supernatant was collected and 500 μL isopropanol 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 removed and 70% ethanol added to the nucleic acid pellet. The pellet was then centrifuged at 15,300×g at 4 °C for 5 min. The supernatant was removed and the nucleic acid pellet dried and dissolved in 200 μL water.
The method for cDNA synthesis was demonstrated from our previous reports^{28 (link),30 (link)}. cDNA was synthesized from the extracted RNA by ReverTra Ace qPCR Master Mix (Toyobo, Osaka, Japan).