Sepasol rna 1 super g solution for rna isolation

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).

RNA was extracted as reported in our previous study^{27 (link)}. 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).

RNA was prepared from cells using the RNeasy Mini or Micro kit (Qiagen, Venlo, Netherlands). To extract RNA from the kidneys, the kidneys 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). Chloroform (200 μL/kidney) was added and centrifuged at 15300 × g at 4°C for 15 min. The supernatant was collected, and 500 μL/kidney isopropanol was added. The sample was mixed well, and the mixture was centrifuged at 15300 × 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 15300 g at 4°C for 5 min. The supernatant was removed, and the nucleic acid pellet was dried and dissolved in 200 μL of water. Complementary DNA (cDNA) was synthesized from the extracted RNA using ReverTra Ace qPCR Master Mix (Toyobo, Osaka, Japan).