Takara sybr green master mix kit

qPCR was conducted following the manufacturer’s instructions. Total RNA was extracted from colon samples and cultured cells using TRIzol reagent. The RNA concentration was determined by the nano spectrophotometer. RNA (1.5 µg) from each sample was subjected to reverse transcription using the PrimeScript Reverse Transcriptase kit (TaKaRa, NO. RR036A) and qPCR analysis performed according to the protocol of TaKaRa SYBR Green Master Mix Kit (TaKaRa, Tokyo, Japan). Primer sequences are presented in Table 1. Gene expression was analyzed via comparative Ct with β-actin as the calibrator and normalized to that of vehicle-treated cells. The fold changes in expression of individual genes were analyzed using the 2^−ΔΔCt method.

To validate the transcriptome data, the relative expression of nine DEGs identified in the transcriptome analysis was assessed by performing qRT-PCR, using three biological and three technical replicates. We performed RNA extraction and cDNA synthesis using the TIANGEN Total RNA Extraction Kit (TIANGEN, Beijing, China) and PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), respectively. The specific primers for selected DEGs were designed using the Primer premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and are shown in (Supplementary Table S1). The actin gene was used as an internal control. qRT-PCR detection was performed on the ABI 7500 Fast Real-Time PCR System using the TaKaRa SYBR Green Master Mix Kit (TaKaRa, Beijing, China).