Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)

cDNA was obtained from total RNA using the PrimeScript™ RT reagent kit with the gDNA Eraser (RR047A; TaKaRa, Dalian, China) according to the standard protocol. The housekeeping gene, eukaryotic elongation factor 1α (PveEF-1α), was used to detect the quality of cDNA [58]. The primers were designed using Primer Premier 6 (http://www.premierbiosoft.com/primerdesign/index.html) and are listed in Additional file 1: Table S1. qRT-PCR was performed by the QuantStudio™ 3 Flex Real-Time PCR System (Thermo Scientific) using SYBR Premix Ex Taq™ II Kit (RR820A; TaKaRa) with three technical replicates. PveEF-1α was considered as the internal control to calculate the relative gene expression level using the ΔΔC_T method.