Hiscript 2 q rt supermix for qpcr gdna wiper kit

Total RNA was extracted using the TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions; RNA quality and concentration were determined by a spectrophotometer. RNA with an OD260/280 ratio between 1.9 and ~2.2 was considered satisfactory and was used for subsequent analysis. For RT-PCR, 1 μg RNA was converted to cDNA with a total reaction volume of 20 μL using the HiScript II Q RT Supermix for qPCR (+gDNA wiper) Kit (Vazyme) according to the manufacturer’s protocol. Reverse transcription was performed using the HiScript II Q RT Supermix for qPCR (+gDNA wiper) Kit (Vazyme). An amount of 1 μg RNA was used in the reverse transcription system for each sample stimulated by V. anguillarum and copper ions in a total reaction volume of 20 μL. In the first step, genomic DNA was removed and the reaction consisted of: 4 μL 4× gDNA wiper Mix, 1 μg RNA, RNase-free ddH₂O added to 16 μL, 42 °C for 2 min; the second step was a reverse transcription system: 5× Hiscript III qRT SuperMix 4 μL with the reaction solution from the first step, 37 °C for 15 min, 85 °C for 5 s. The product was used immediately for subsequent experiments.

Total RNA extraction was performed using the TransZol kit (TransGen Biotech, Inc., Beijing, China), and cDNA synthesis was carried out using the HiScript^® II QRT SuperMix for qPCR (+gDNA wiper) kit from Vazyme (Piscataway, NJ, USA). The LightCycle * 96 Real-Time PCR System from Roche (Basel, Switzerland) was employed for qRT-PCR experiments. A 50 μL reaction system was configured, and each sample underwent three biological repeats with three technical repeats. The HiScript^® II Q RT SuperMix for qPCR (+gDNA wiper) kit from Vazyme (Piscataway, NJ, USA) was utilized for reverse transcription. Relative expressions were calculated using the 2^−ΔΔCt formula. Primers for VfBBXs and the actin EF1α gene are detailed in Table S3. All data are expressed as mean ± SDs (Student’s t-test; * p < 0.05, ** p < 0.01, and *** p < 0.001 considered statistically significant).

Total RNA extracted from the indicated mycelia or fruiting bodies using a spin column fungal total RNA purification kit (Sangon Biotech) was used to synthesize first-strand cDNA using a HiScript II Q RT Supermix for qPCR kit (+ gDNA wiper) (Vazyme), and the resulting cDNA was used for qRT-PCR analysis using a pair of specific primers for each gene (see Table S2) and AceQ qPCR SYBR green Master Mix (Vazyme) (20 (link)). The gene expression levels were normalized to β-tubulin, and the fold expression of target genes relative to β-tubulin was calculated according to the 2^−ΔCT method as follows: −ΔC_T = −(C_T target − C_{T β-tubulin}).