Wheat Seedling Gene Expression Analysis

Total RNA was extracted from 25-day-old wheat seedlings using the GeneJET Plant RNA Purification Kit (Thermo Scientific^TM). Approximately 2 μg of total DNase-treated-RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit by Invitrogen (Karlsruhe, Germany). Expression analysis of ET and polyamine biosynthesis genes in wheat was performed by using primers (Table 1), designed through primer 3.0 (Untergasser et al., 2012 (link)). TaACTIN2 (AB181991) served as an internal control, previously reported by Xu et al. (2013) (link). Amplification of each gene was performed in triplicate by using an ABI PRISM 7900HT sequence detection system (Applied Biosystems), and amplification products were visualized using SYBR Green (Applied Biosystems). Amplification curves were analyzed with a normalized reporter (Rn: the ratio of the fluorescence emission intensity of SYBR Green to the fluorescence signal of the passive reference dye). RT-qPCR expression analysis was performed by using three independent biological replicates with at least three technical replicates.