Primerscript first stand cdna synthesis kit

The RNA was extracted from seven tissues, namely, the intestine, liver, peripheral blood leukocytes (PBLs), muscle, head kidney, spleen, and gills using Trizol Reagent (Invitrogen, USA). The first-strand synthesis used the Primerscript™ First Stand cDNA Synthesis kit (Takara, China). RT-qPCR was performed using the LightCycler^® 480 II Real-Time System (Roche, Switzerland) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The primers and housekeeping genes are listed in Table 1. Briefly, the cDNA was adjusted to 400 ng/ml. Each reaction consists of 10 μl of 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme), 0.4 μl of forward and 0.4 μl of reverse primers (10 μM), 2 μl of cDNA, and 7.2 μl of ddH₂O. The thermal cycling profile consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, and extension at 60°C for 30 s. An additional temperature ramping step was utilized to produce melting curves of the reaction from 60°C to 95°C. The β-actin gene was used as an internal control. The 2^−ΔΔCT method was used to analyze the expression level of the CD80/86 gene.

Total RNA of tea leaves was extracted using a rapid RNA isolation kit (Tiangen Biotech, Co., Ltd., Beijing, China). Then, cDNA was synthesized using PrimerScript First Stand cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was performed according to Zhu et al., 2019 [17 (link)]. SYBR Premix Ex Taq (TaKaRa, Dalian, China) was used for relative expression analysis and data was performed by the 2^−ΔCT method. CsGAPDH (TEA025584.1) was used as an internal reference. All primers were designed with Primer 5.0 software and primer sequences are shown in Table S3. The procedure of qPCR was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s.