Quantitative RT-PCR Gene Expression Analysis

Six functionally important and representative genes were selected for validation of gene regulation using quantitative RT-PCR. Gene-specific primers were designed for each gene, and the ADH3 gene was used as the internal control [25 (link)]. Total RNAs of peanut leaves were extracted using E.Z.N.A. Plant RNA Kit (Omega Bio-tek, USA, R6827-01). Samples of 1 μg RNA were used to synthesize the first-strand cDNA using a reverse-transcription system (Promega, USA, A3500) according to the manufacturer’s instructions. Quantitative real-time PCR was performed on the Eppendorf Realplex (4) PCR machine (Eppendorf, Hamburg, Germany) using a reaction volume of 20 μl contained 2× SYBR Green PCR RealMaster Mix (TianGen, FP202-02), forward and reverse primers, and cDNA template (diluted 1:40). All samples were run in duplicate. The real-time PCR reaction was run for one cycle (94°C for 3 min) followed by 40 cycles (94°C for 15 s, 59°C for 20 s, 68°C for 40 s) and fluorescence emissions were measured after each of the repetitive cycles. The results were obtained based on the average of three parallel experiments.