Seasonal Expression Analysis of Phoebe sinensis

Total RNA used for RT-qPCR analysis was prepared from the P. sinensis testes in April, July, and October with three biological replicates. The cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Primer sequences were designed by using Beacon Designer 7.0 (Premier Biosoft International, USA) software (Supplementary Table 1). The RT-qPCR analysis with three technological replications was performed on an iCycler Real-Time PCR Detection System (Bio-Rad, USA) according to a previous report (Kim et al., 2013 (link)). The β-Actin gene was used as the internal control. The relative expression levels of the genes were calculated using the delta-delta-CT method.