4.6. Validation of RNA-Seq Data by qRT-PCR

Fifteen DEGs were selected to test the reliability of the RNA-Seq data by qRT-PCR with three biological replicates. Gene-specific primers were designed using Primer Premier 5.0 (see Supplementary Material Table S1) and synthesized by Invitrogen Trading Co., Ltd. (Shanghai, China). Total RNA extraction, cDNA synthesis, qRT-PCR, and statistical analyses were performed as previously described [51]. Amplification was conducted using SYBR Premix Ex Taq™ (Toyobo Co., Ltd., Osaka, Japan). The RT-PCR program was as follows: initial denaturationat 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. Relative quantification of gene expression levels was performed using Actin 11 as an internal reference gene for normalization. The comparative threshold cycle (Ct) 2^−ΔΔCt method was used to calculate fold changes in gene expression [52].