Hiscript rt supermix for qpcr

Total RNA was extracted using the RNA extraction Kit (Fastagen, 220,010). Reverse transcription was performed using cDNA Synthesis Kit (Vazyme, R312-01). The reverse transcription conditions were 37°C for 15 min and 85°C for 5 s. The RT-PCR were performed using HiScript RT superMix for qPCR (Vazyme, R122-01) and the reaction conditions were initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. Primers and reagents used were as follows: Fhl2(rat), Forward: 5′- TCTGACCCCACAGGTTGCTG-3′; Reverse: 5′- TCACAGGTGTTGGCATAGAGC-3′. Cfh(rat), Forward: 5′- GTGTAAAGCCCCGAAGTCAAC-3′; Reverse: 5′- GGAGGGCAGAATCTTTTCTCATT-3′. Acta2 (rat), Forward: 5′- GTGTTCAGAGAGGGTGAGCC-3′; Reverse: 5′- TCAGGTTGGTCCTCTGGTCT-3′. Gapdh (rat), Forward: 5′- GCATCTTCTTGTGCAGTGCC-3′; Reverse: 5′- GATGGTGATGGGTTTCCCGT-3′.

The total RNA from leaves and flowers was extracted separately using a FastPure Plant Total RNA Isolation Kit (Polysaccharides and Polyphenolics-rich) (Vazyme, RC401-01, Nanjing, China) following the manufacturer’s instructions. The RNA concentrations were measured with a NanoDrop 2000 spectrophotometer. High-quality RNA (1 μg) from each sample was reverse-transcribed using the HiScript RT SuperMix for qPCR (Vazyme, R323-01, Nanjing, China) following the protocol from the manufacturer. Next, qRT-PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02, Nanjing, China). RcActin was used as the internal control gene. The relative expression levels of genes were calculated via the 2^−ΔΔCT method. Three biological replicates and three technical replicates were performed for each experiment. The primer sequences are listed in Table S1.

qRT-PCR was used to assess the influence of carvacrol on gene expression of different virulence. A. hydrophila was treated with carvacrol (0, 1/4 MIC) for 20 h, and 1% DMSO was used as a negative control. Total RNA was extracted following the guidance and instruction of the RNAiso Plus kit (Takara, Daling, China). RNA quantities and concentrations were determined using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Double-stranded cDNA was synthesized using Hiscript RT supermix for qPCR with a gDNA wiper (Vazyme, Nanjing, China). Real-time PCR was performed using SYBR green real-time PCR mix (Bio-Rad) on a CFX real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The mRNA expression of targeted genes (flaB, aha, ompA, ahp, act, aerA, hly, ela, and AhyR) was normalized to the internal control (rpoB gene). Each assay was performed in triplicate. The gene-specific primers used in this study are listed in Table S1.

Using the FastPure Cell/Tissue Total RNA Isolation Kit, total RNA was isolated from the cells (Vazyme, Nanjing, China). Reverse transcription was carried out using HiScript RT supermix for qPCR (Vazyme, Nanjing, China). The expression levels of the genes were assessed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) via ABI 7500 Fast Real-Time System. The reaction mixtures underwent 35 cycles of 95° C for 15 seconds, 60° C for 30 seconds, and 95° C for 15 seconds after being incubated at 95° C for 2 minutes. The relative expression of mRNA was normalized using the 2-^ΔΔCt method relative to GAPDH. Primer sequences were as follows: ANGPTL4, 5’-GTCCACCGACCTCCCGTTA-3’ (forward) and 5’-CCTCATGGTCTAGGTGCTTGT-3’ (reverse); CDKN3, 5’-TCCGGGGCAATACAGACCAT-3’ (forward) and 5’-GCAGCTAATTTGTCCCGAAACTC-3’ (reverse); PLK1, 5’-CCTGCACCGAAACCGAGTTAT-3’ (forward) and 5’-CCGTCATATTCGACTTTGGTTGC-3’ (reverse); SLC2A1, 5’-TCTGGCATCAACGCTGTCTTC-3’ (forward) and 5’-CGATACCGGAGCCAATGGT-3’ (reverse); GAPDH, 5’-GATCATCAGCAATGCCTCCT-3’ (forward), reverse: 5’-TTCAGCTCAGGGATGACCTT-3’ (reverse).