Platinum sybr green rt qpcr mix

To detect the transcript levels of PsV5 during infection of wheat Suwon11 by Pst race CYR31, wheat leaves infected with urediniospores were collected at 6, 12, 18, 24, 36, 48, 72, 120, 168, 196, 216, and 256 hpi. To evaluate the efficiency of BSMV-mediated HIGS, the fourth leaves of PsV5-silenced plants were sampled at 24, 48, and 120 hpi with Pst race CYR31. RNA was extracted from all samples using the Quick RNA Isolation Kit (Huayueyang Biotechnology, China, Beijing). For cDNA synthesis, 3.0 μg of RNA was used with the RevertAid First Strand cDNA Synthesis Kit (MNI, K1622) using the RT–PCR system and oligo(dT)18 primer. PstEF1 and TaEF1 were chosen as internal control genes to normalize RNA levels in Pst and wheat leaves, respectively. Quantitative real-time PCR was performed with SYBR Green fluorescence and a ROX reference dye (Platinum SYBR Green RT-qPCR Mix, Thermo Fisher Scientific) on an ABI PRISM 7300 sequence detection system (Thermo Fisher Scientific) with the primers listed in Supplemental Table 3. Raw data were converted into expression data by the 2^−ΔΔCt method. Each sample was analyzed as three biological replicates, and each PCR analysis included three technical replicates.