Expressional validation of carotenoid biosynthesis genes with qRT-PCR

The expression levels of the seven unigenes that participate in the MEP pathway were examined by qRT-PCR. The primers for qRT-PCR are shown in Table 2. The expression levels were measured with a Baiyuan ASA-4800 Real Time PCR System using SYBR Green fluorescent dye (Takara, China) according to the manufacturer’s instructions. The cycling profile included a step at 95 °C for 30 s, followed by 40 cycles of amplification (95 °C for 5 s and 60 °C for 34 s). The relative gene expression was calculated using the 2^-ΔΔCt relative quantitative method. All MEP pathway genes were chosen for analysis of expression patterns in different samples that were subjected to the three different temperatures (12 °C, 20 °C, and 28 °C). The expression levels of these selected genes from qRT-PCR analyses were assessed in comparison to the differentially expressed genes (DEGs) from RNA-Seq.

Design primers for Real-Time PCR