SYBR Green qPCR confirmation of DGAT gene expression in tung tree tissues.

(A) SYBR Green qPCR primer optimization for DGAT2. The qPCR reaction mixtures contained 5 ng RNA-equivalent cDNA from tung seeds, leaves and flowers, various concentrations of the forward primer and reverse primer and iQ SYBR Green Supermix. (B) SYBR Green qPCR efficiency. The qPCR reaction mixtures contained variable concentrations of RNA-equivalent cDNA from tung seeds, the optimized concentrations of each primer and probe (200 nM) and iQ SYBR Green Supermix. The results using RNA isolated from stage 4 of tree 1 are shown in the figure. (C) Melt curve analysis of SYBR Green qPCR assays. The qPCR reactions contained 5 ng RNA-equivalent cDNA from tung seeds (left), leaves (middle) and flowers (right), the forward primer, reverse primer and iQ SYBR Green Supermix. (D) Gel electrophoresis of SYBR Green qPCR amplification products. The qPCR products after 40 cycles of amplification were separated by electrophoresis with 3% agarose gel at 100 V for 30 min. T1.5 and T1.6 represent RNA extracted from seed stages 5 and 6 of tung tree 1. Similar gel electrophoresis results were obtained using other stages of tung seeds (data not shown). Lanes 1–6 represent qPCR products for Rpl19b, Gapdh, Ubl, Dgat1, Dgat2, and Dgat3, respectively. The lane “Ladder 100 bp” represents the 100 bp DNA ladders with the lowest band being 100 bp. (E) Relative DGAT mRNA levels in tung seeds, leaves and flowers. The qPCR reaction mixtures contained 5 ng RNA-equivalent cDNA from tung seeds, leaves and flowers and the optimized concentrations of each primer (200 nM). The means of two determinations of DGAT mRNA levels are presented. T1 represents RNA extracted from tung tree 1. The number after T1 represents the week when tung seeds were collected.