Quantitative Real-Time PCR Analysis of Gene Expression

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.