Hiscript 2 qrt supermix for qpcr reverse transcription kit

Total RNA was isolated from brain tissue or cultured brain cells using Trizol Reagent (TaKaRa, Shiga, Japan). The extracted RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water to prevent contamination. Then, a multi-plate reader (BioTek, Winooski, VT, USA) was used to detect the concentration of RNA to further determine whether protein contamination or degradation of RNA existed. One microgram of total RNA was reverse transcribed into complementary DNA with Hiscript II Q RT SuperMix for qPCR Reverse Transcription Kit according to the manufacturer’s instructions (Vazyme, Nanjing, China). Quantitative real-time PCR was performed with AceQ^® qPCR SYBR^® Green Master Mix (Vazyme, Nanjing, China) using the MyiQTM 2 double-color real-time PCR detection system (Bio-Rad, Hercules, CA, USA). In the PCR protocol, except the biological replicates involved in the experiment, 3 technical replicates were set up to reduce the impact of experimental operations. In this study, we used one effective reference gene (RPL13A), which was identified to exhibit high transcriptional stability between different treatments in mandarin fish (Siniperca chuatsi). The relative expression level of the target gene was quantified by the 2^−ΔΔCt calculation method. Based on the sequences acquired in the Chinese Perch Genome Database [55 (link)], primers used for real-time PCR are listed in Table 1.

The chemicals utilized in this study were as follows: dextran sulfate sodium (DSS; MP Biomedicals, LLC, Solon, OH, USA; lot number: BJ14745, molecular weight: 5000); berberine hydrochloride (BBR; Shanghai Yuanye Bio-technology Co., Ltd., Shanghai, China; lot number: Y18D8C50814, molecular weight: 371.81); glucose-regulated protein 78 (GRP78), caspase-12, and caspase-3 primary antibodies (Abcam, Cambridge, UK; lot numbers: GR309483-1, ab62484, and ab13847, respectively); anti-rabbit and anti-mouse secondary antibody (Beijing ZSGB-BIO, Beijing, China; lot numbers: K167722B and K175212C, respectively); proteinase K (Beijing Tiangen, Beijing, China; lot number: M2011); DAB chromogenic reagent kit (Beijing ZSGB-BIO, lot number: K167722A); TUNEL in situ apoptosis detection kit (Roche, Basel, Switzerland; lot number: 11906800); high-purity total RNA rapid extraction kit (Shanghai Generay, Shanghai, China; lot number: 1703G01); HiScript-II Q RT SuperMix for qPCR reverse transcription kit (Nanjing Vazyme, Nanjing, China; lot number: 7E092G6); and ChamQ SYBR Color qPCR Master Mix (Nanjing Vazyme Co.; lot number: 7E092H6).

Total RNA was isolated using TRizol (#R401-01-AA, Vazyme Biotech) following the manufacturer's protocol. RNAs were then reverse transcribed using HiScript II Q RT SuperMix for qPCR Reverse Transcription Kit (#R223-01, Vazyme Biotech) following the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) was performed using AceQ® Universal SYBR® qPCR Master Mix (#Q511, Vazyme Biotech). Primers were synthesized by Integrated DNA Technologies. Target gene expression was normalized to the GAPDH reference gene and relative expression levels were determined using the 2^−ΔΔCt method. The primers used in qRT-PCR were listed in Supplementary Table 13.