Two color real time pcr detection system

Total RNA was extracted using the TRizol reagent. First-stand cDNA was synthesized by a PrimeScript First-strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Real-time PCR was performed in a Two-color Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Mix (TaKaRa, Dalian, China). The 18S rRNA and Actin were chosen as internal controls in sugar beet and Arabidopsis, respectively [36 (link)]. PCR reaction was carried out in 10 µL volumes using the following amplification protocol: 94 °C for 4 min; 94 °C for 30 s, 53 °C for 20 s, and 72 °C for 70 s; and 72 °C for 4 min and 45 cycles. The primers used for qRT-PCR analysis are listed in Table S1. The primer sequence of SOD and POD genes for qRT-PCR were acquired from reference [30 (link)], and other primers were designed by Primer 5. A total of three biological replicates and three technical replicates were performed for the quantitative Real-Time PCR analyses.

Total RNA for RNA-seq samples was also used for qRT-PCR in this study. Reverse transcription into cDNA was performed with SuperReal PreMix Plus (TIANGEN Biotech, Beijing, China). All of the experiments were performed following the protocols included with the kits. Primers were designed using Primer Premier 6. The qRT-PCR was performed using a MiniOpticon Two-Color Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). The PCR conditions were 95 °C for 30 s, followed by 45 cycles of 95 °C for 3 s for denaturation and 60 °C for 30 s for annealing and extension. The relative expression levels of the target genes were normalized and then calculated using the comparative Ct (2^−∆∆Ct) method. For each sample, PCR was repeated three times. EF1 (elongation factor 1) was selected as the internal control for normalizing the results, and the FER ovules in FNM1 stage were used as a reference sample whose value was set to 1. The relative gene expression was evaluated using the comparative cycle threshold method.

Arabidopsis mutants clf-29 (SALK_N521003)[71 (link)], swn-21[72 ], lhp1-6 (SALK_011762)[57 ], tfl2-2 (CS3797)[73 (link)], atring1a,b[13 (link)] and atbmi1a,b[12 (link),16 (link)] in Col-0 background have been described previously. All plants except clf-29swn-21 were grown in soil under long day (16 hour) photoperiods at 22°C in green house, and seedlings were harvested after 2 weeks. clf-29swn-21 was grown on half-strength Murashige and Skoog (MS) medium in the same long day conditions as above, and the whole plants were harvested after 2 weeks. Harvested materials were frozen in liquid nitrogen for total RNA isolation or directly vacuum-infiltrated with formaldehyde crosslinking solution for ChIP assay. Quantitative real-time PCR analysis of ChIPed DNA with specific primers (S8 Table) was performed on the two-color real-time PCR detection system (BIO-RAD, Hercules, CA, USA) using the SYBR Green Realtime PCR Master mix (TOYOBO) to represent the relative methylation levels.