Trizol plus rna puri cation kit

A TRIzol Plus RNA puri cation kit (Invitrogen Canada Inc.) was used to isolate RNA from 80 mg of the ground jejunum. The quantity and quality of the isolated RNA were evaluated using a Nanodrop 2000 spectrophotometer (ThermoFisher Scienti c). The integrity of the total RNA was con rmed by agarose gel electrophoresis. A total of 2 μg of total RNA was used to synthesize cDNA using a high-capacity cDNA synthesis kit (Applied Biosystems) following the supplier's protocol. Quantitative real-time PCR was performed in duplicate reactions, using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories), as described by Waititu et al. (Waititu et al.) . For qPCR ampli cation, negative controls were prepared by replacing the cDNA with nuclease-free water. A melt curve analysis was performed with a temperature gradient of 0.1°C/s from 70°C to 95°C to con rm that only speci c products were ampli ed. Pairs of primers for each gene were designed and checked for target identity using the National Center for Biotechnology Information database (Supplemental Table 1). Each set of primers used for qPCR ampli cation con rmed that their e ciencies were between 90% and 110%.