After the MC1 and hpt genes were confirmed in transgenic rice, expression of MC1 was analyzed using real-time PCR at both T0 and T1 generations and at seedling and tillering stages. Total RNA extraction was done with the method of Liu et al. [27 (link)] with slight modifications and cDNA was synthesized using Thermo Fischer Revert-Aid First Strand cDNA Synthesis Kit K-1622. The analysis was performed on a Bio-Rad Real-Time PCR system, using the transformed cDNA as templates and MC1 primers to analyze the gene. The reference gene was actin (ACT 1) with to following primer sequence:
To quantify the amount of dsDNA, One-Step SYBR Green Master Mix was used. The thermal setting was denaturation at 95°C for 3 minutes, followed by 39 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds and extension at 72°C for 30 seconds. The qPCR samples were run in sets of three. Actin was utilized as an internal control for data standardization. Comparative quantification of gene expression was analyzed using the delta Ct method in tested samples and the standard deviation was calculated [28 (link)]. The graphs were plotted for the relative expression of MC1 gene compared to the internal control.
To quantify the amount of dsDNA, One-Step SYBR Green Master Mix was used. The thermal setting was denaturation at 95°C for 3 minutes, followed by 39 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds and extension at 72°C for 30 seconds. The qPCR samples were run in sets of three. Actin was utilized as an internal control for data standardization. Comparative quantification of gene expression was analyzed using the delta Ct method in tested samples and the standard deviation was calculated [28 (link)]. The graphs were plotted for the relative expression of MC1 gene compared to the internal control.