The remaining RNA samples used in the RNA-seq experiment were used for the synthesis of first-strand cDNA according to Li et al.’s method [8]. Gene expression analysis was executed by real-time PCR as previously described [50]. The housekeeping genes EF1 and UBCE2 were used as endogenous references for normalization [51]. Specific primers were designed by primer 3.0 (Addition file 10). The amplification specificity of all target and reference gene was confirmed by observing a single dissociation curve for each pair of primers. The data of similar amplification efficiencies between the target genes and reference genes were used for subsequent analyses. The ΔCt method was used to calculate the values of the target gene relative to the reference gene [52]. Data are calculated as the mean ± standard deviation (SD) of three replications performed in 96-well plates. The data were analysed using CFX Manager™ v3.0.

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