Sybr premix kit

Liquid nitrogen was used to smash frozen samples of leaves. After that, total RNA was extracted with the use of a commercial RNA extraction kit from AidLab (China) and reverse transcribed with a commercial kit (TaKaRa, Japan). The obtained cDNA was put through a qRT-PCR analysis utilizing a Quantagene Real-Time System q225 (Novogene, China) and a SYBR Premix kit (Bio-Rad, USA). The following served as the procedures: a pre-denaturation period of 5 minutes at 95°C is followed by 40 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 30 seconds at 56°C. Each 12-uL reaction included 6 uL of SYBR Green PCR mix, 0.5 uL of primer, and 100 ng of cDNA. The primer pairs F_RT-CsBZIP40/R_RT-CsBZIP40, F_RT-CsWRKY43/R_RT-CsWRKY43, F_RT-CsPrx53/R_RT-CsPrx53, and F_RT-CsSOD13/R_RT-CsSOD13 (Table S5, see online supplementary material) were utilized for these assays. The 2^-∆∆CT approach was used to establish relative gene expression [43 (link)].

For quantitative RT-PCR, frozen tissue was ground in liquid nitrogen and then total RNA extracted from leaf samples using a Miniprep kit purchased from AidLab Ltd. (Beijing, China). The RNA was then reverse transcribed to cDNA using a kit from TaKaRa (Dalian, China). A SYBR Premix kit (Bio-Rad, USA) and the Quant Studio 7 system (Applied Biosystems, USA) were used for qRT-PCR, with actin from citrus (GenBank accession: GU911361.1) used as the internal control gene, amplified using primers F-actin (CATCCCTCAGCACCTTCC) and R-actin (CCAACCTTAGCACTTCTCC). qRT-PCR was performed by heating samples to 95°C pre-denaturation for 5 min, followed by 40 cycles at 95°C for denaturation for 10 s, and 56°C for annealing and extension for 30 s. The 20 μL reaction mixture comprised 100 ng cDNA, 0.5 μM primers and 10 μL SYBR Green PCR mix. The 2^-ΔΔCT method was used to evaluate relative expression levels of each gene investigated [37 (link)]. Specific primers for CsPR1, CsPR5, CsNPR1 and CsICS were designed by NCBI Primer Blast program and are listed in S1 Table. For each gene, three biological and three technical replicates were used for the qRT-PCR assay.