Qrt pcr primers

Total RNA from was isolated using RNeasy Plus Universal Mini Kit (73404, Qiagen, Valencia, CA) including DNase treatment. Diluted by 10 mM Tris buffer, RNA concentration and purity of the samples were measured by use of spectrophotometer (Beckman Coulter, Brea, CA). Afterwards, following the standard one-step RT-PCR protocol for use with the QuantiTect SYBR Green RT-PCR Kit (204243, Qiagen) and Mx3005P QPCR System (Agilent Technologies Inc., Santa Clara, CA), template RNA (10 ng/reaction) was added to prepare reaction mix. The one-step PCR cycling conditions were: 50°C for 30 minutes, 95°C for 15 minutes, 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. S9 was employed as a housekeeping gene. Data were collected and analyzed by MxPor QPCR software (Agilent Technologies Inc.); relative gene expression is calculated as a ratio of Ct of the interested gene to that of S9. All qRT-PCR primers were purchased from Qiagen.

Total RNA was prepared from the jejunum using Trizol (Invitrogen) and then transformed to cDNA using a reverse transcription kit (Qiagen, Hilden, Germany). As mentioned in Alkhudhayri et al. (2020) , the quantitative real-time PCR (qRT-PCR) was conducted. Both MUC2 and MUC4 mRNA expression were determined using the commercially obtained Qiagen qRT-PCR primers. The levels of mRNA had been normalized to 18S rRNA.