Abi7500 steponeplus real time pcr system

For the expression study of flavonoid biosynthetic and antioxidative pathway genes under normal and salt stress conditions, a quantitative real-time PCR was performed using 4.5 µl of diluted cDNA with the DyNamo Flash SYBR Green qPCR Kit (Thermo Scientific) and gene-specific primers (Supplementary Table S2) designed by PrimerExpress 3.0.1 software (Applied Biosystems, USA) to a final volume of 32 µl for each reaction. The expression analysis was carried out in triplicate by SYBR Green dye chemistry detection with an ABI 7500 Step One Plus Real-Time PCR System (Applied Biosystems). The initial stage was 94°C for 5min, followed by 45 cycling stages of 94°C for 30 s, 58–59°C for 30 s, and 72°C for 30 s (data collection), with a step and hold melt curve stage. The experiments were analysed according to baseline and a manual threshold, and data was collected with ABI 7500 System Step One v2.2.2 Software. Relative quantifications were calculated using the Ct method (comparative 2^ddCT method) and normalized with the expression level of an endogenous gene 26SrRNA.

Primers specific for the chosen gene sequences were designed using Primer Premier (version 5.00) software (Supplementary Table 1). Three commonly used internal reference genes (UBI, GAPDH, and 18S rRNA) were selected as candidate internal reference genes, and the Ct values of candidate internal reference genes were obtained and analyzed by geNorm, NormFinder, and BestKeeper (Vandesompele et al., 2002 (link); Andersen et al., 2004 (link); Pfaffl et al., 2004 (link)) to select the best internal reference genes. Vazyme miRNA Design (v1.01) software was used to design primers for the real-time fluorescence quantification of miRNAs, and primers for first-strand cDNA synthesis were synthesized using the stem-loop method. The designed primers are shown in Supplementary Table 1.
The C. fortunei reverse transcription cDNA products and the first-strand cDNA products were diluted 10 times, and 3 replicates were analyzed for each sample and internal reference. Then, according to the kit’s protocol, qPCR was carried out on an ABI 7500 Step OnePlus Real-time PCR System (Applied Biosystems, Foster City, CA, United States), with the following conditions: 95°C for 20s, 95°C for 10s, 95°C for 10s, and 60°C for 34s.