2.6. Differentially expressed gene validation via qRT-PCR
Ten differentially expressed genes (DEGs) were randomly selected to verify their differential expression. The qRT-PCR primers were designed using Primer 6 and are listed in Table 1. The actin gene was used as the control. The qRT-PCR was carried out on a real-time PCR system (Eppendorf, Hamburg, Germany). Reacted mixture consisted of 1 μl cDNA (60 ng/μl), 10 μl Bestar SYBR Green qPCR Mastermix (DBI, Bioscience Inc., Hamburg, Germany), 1 μl of each primer, and 7 μl mill i-Q water. Reactions were performed at 95 °C for 30 s; 30 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 60 s. The qRT-PCR results were obtained from three biological replicates, and the gene relative expression levels were calculated using the 2−ΔΔCt method.
Table 1. Sequences of primers used for gene differential expression analysis