Chamq sybr color qpcr master mix kit

RT‐qPCR was conducted as previously reported.^[51 (link)
^] Briefly, TRIzol reagent was used to extract RNA from the kidney samples, and cDNA was synthesized using the HiScript II Reverse Transcription System (Vazyme, Nanjing, China). RT‐qPCR analyses were conducted using the ChamQ SYBR Color qPCR Master Mix kit (Vazyme) on a Bio‐Rad Cycler system (Bio‐Rad, Hercules, CA, USA). The primer sequences are given in Table S2 (Supporting Information).

The cDNA obtained by reverse transcription was carried out qRT-PCR as a pre-experiment. According to the Ct values obtained in the pre-experiment, 2 μL cDNA was then taken, and the Ct value of cDNA was diluted to 20 μL with dd water as the optimal cDNA concentration required for the qRT-PCR reaction of different target genes. The software Primer Premier 5.0 was used to design qRT-PCR primers. The primers used in the experiment were listed in Table S2. The qRT-PCR reaction mixture was mixed using a ChamQ SYBR Color qPCR Master Mix Kit (Vazyme, Q431-02, China), which was 20 μL including 10 μL 2× ChamQ SYBR Color qPCR Master Mix, 0.4 μL forward and reverse primer of the target gene, respectively, 8.2 μL DEPC-treated water and 1 μL cDNA. The pre-denaturation temperature of PCR amplification was 95 °C for 5 min, followed by 40 cycles (95 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s), and then the determination of the melting curve (95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s). The β-actin gene was used as a reference gene for the qRT-PCR.

Total RNA of the hippocampus and cortex was extracted using RNazol following the manufacturer’s instructions (Takara, Dalian, China): the concentration of RNA was determined spectrophotometrically at 260 nm. The quality of RNA was further assessed by the ratio of absorbance at 260 and 280 nm. The values of A260/A280 from 1.9 to 2.1 were considered as reasonable. Total RNA (1.5 μg) was applied to synthesize cDNA using HiScript-II Q RT SuperMix for qPCR kit (Vazyme Biotech, Nanjing, China) following the manufacturer’s manual, and RT-PCR was performed using reagents and protocols from ChamQ SYBR Color qPCR Master Mix kit (Vazyme Biotech). The sequences of primers were shown: 5’-GCC TCC TCT CCT ACT TCG G-3′ (sense primer, S) and 5′-TCA GCC CAT CTT CTT CCA G-3′ (antisense primer, AS) for Bax; 5′- AAA CCC TCC ATC CTG TCC-3′ (S) and 5′-TCC TAA ACC CTG CTT CCC-3′ (AS) for murine Bcl-2; 5′-GAA GCA GGC ATC TGA GGG-3′ (S) and 5′-AAG GTG GAA GAG TGG GAG TT-3′(AS) for murine GAPDH. The relative expressions of Bax and Bcl-2 mRNA were normalized to the amount of GAPDH in the same cDNA following the relative quantification method (2^-ΔΔCT method).

The expression of stress-related flavonoid-related genes was measured by qRT-PCR in WT and transgenic lines of A. thaliana or apple calli. Total RNA was extracted from A. thaliana leaves or apple calli using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized using the PrimeScript™II 1st Strand cDNA Synthesis Kit (Clontech TaKaRa, Beijing, China). Quantitative RT-PCR was conducted using the ChamQ SYBR Color qPCR Master Mix Kit (Vazyme, Shanghai, China) with a QuantStudio™ 5 Real-Time PCR System. Each reaction was performed in triplicate, and data were analyzed as previously described [55 (link)]. Atactin1 (At2g37620) or MdActin (XM_029088423.1) was used as an internal control. All the quantitative PCR primers are listed in Table S1.
In addition, the expression level of mature miR156 was confirmed by stem-loop RT-PCR as described [56 (link)]. The cDNA synthesis was performed using the prime script first strand cDNA Synthesis Kit (Takara, China), according to the manufacturer’s instructions, using a stem-loop RT primer instead of an oligo (dT) primer (Table S1). Subsequently, PCR was performed to detect the expression level of miR156 using the miR156-specific forward primer and the stem-loop-specific reverse primer (Table S1). The apple 5.8S rRNA (GenBank accession no. AF186480) was used as a reference gene.

Gene expression was analyzed by CFX96 Touch Real-Time PCR Detection System (Bio-rad, CFX96 touch). Total RNA was extracted from the leaves, stems, skin, flesh, calli, and tissue culture seedlings of apples using the TIANGEN Plant RNA Extraction Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript^TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Kusatsu, Japan) according to the manufacturer’s instructions. The cDNA was serially diluted 10 times and stored at −20 °C. qRT-PCR was performed using the ChamQ SYBR Color qPCR Master Mix Kit (Vazyme, Nanjing, China) using 1 μL of the template cDNA and 0.5 μL of each gene-specific primer (Supplementary Table S1). The NCBI Primer-BLAST tool was used to design the primers. The MdActin gene (XM_029088423.1) was used as an internal reference. The relative expression levels of the target genes were determined using the 2^−ΔΔCT method, as described by Livak and Schmittgen [61 (link)]. The specificity of each gene was determined using a dissociation curve analysis. All experiments were repeated with three biological replicates and three technical replicates. The gene-specific primer sequences are shown in Supplemental Table S1.

DEGs were randomly selected for quantitative real-time PCR (qRT-PCR) to validate the quality of the sequencing data. The selected genes were extracted from the B. dothidea genome (ASM1150312v2), and the special primers were designed at the Primer Premier 5.0 software (Table 1). The cDNA was synthesized using the HiScript^® lll RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme Biotech, Nanjing, CN). According to the instructions of the ChamQ™ SYBR Color qPCR Master Mix kit (Vazyme Biotech, Nanjing, CN), the expressions of selected genes were analyzed by ABI7500 thermal cycler (Applied Biosystems, CA). The total reaction system was 10 μl, including 5 μl of 2 × ChamQ SYBR Color qPCR Master Mix, 0.2 μl of each primer, 0.2 μl of the 50 × ROX Reference Dye I, 1 μl of cDNA, and 3.4 μl of the ddH₂O. The reaction conditions were following: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s and 60°C for 30 s, then 72°C for 30 s, and a final extension at 72°C for 10 min. Actin was used as the internal reference gene, and the relative expression was calculated by 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). Three biological replicates were set up in the experiment.