Comprehensive RNA Analysis Pipeline

RNA extraction, complementary DNA (cDNA) synthesis, and RT-qPCR were conducted in accordance with our previous studies [76 (link)]. Briefly, cDNA was made using the SuperiorScript III cDNA synthesis kit (#EZ405S, Enzynomics, Daejeon, Republic of Korea). The cDNA was diluted with RNase-free water to a final concentration of 8 ng/µL, and the samples were kept at −80 °C. RT-qPCR was carried out using TOPreal^TM SYBR Green qPCR PreMix (#RT500M, Enzynomics, Daejeon, Republic of Korea) and the LineGene 9600 Plus machine (BIOER, Hangzhou, China) following the manufacturer’s instructions. The primers for RT-qPCR are shown in Table 1. The annealing temperature for the reaction was 58 °C, and the built-in software created the amplification curves and calculated the threshold cycle values. The GAPDH reference gene was used to normalize all the readouts. Data were reported as the mean relative values compared to the CON group using the 2^−∆∆CT method.