RNA Isolation from Mouse Intestinal Crypts

As described in previous study [45 (link)], the isolated crypts were cracked by TRizol (Takara, Osaka, Japan) on ice for 10 min, the chloroform was added into an upside-down tube and mixed, centrifuged at 4 °C at 12,000 rpm for 15 min. The upper water phase was transferred to a new tube, and the same volume of isopropyl alcohol was added and mixed, precipitated for 2 h at −20 °C, and centrifuged for 10 min at 12,000 rpm at 4 °C. The white precipitate at the bottom of the tube was RNA. The RNA was washed twice with 75% ice ethanol and dissolved in enzyme-free water after air drying in the ventilation cabinet. Nanodrop 2000 was used to measure the concentration and purity of RNA, the performed reverse transcription (Takara, Osaka, Japan), and qRT-PCR (Takara, Osaka, Japan) according to the kit instructions. A total of 30 experimental animals C57BL/6 mice were used.