Quantitative PCR Analysis of Gene Expression

RNA from the same libraries was used for quantitative PCR analyses (Livak and Schmittgen 2001 (link); Liu et al. 2015a (link)). Primers designed with Primer Premier 5.0 are shown in Additional file 1: Table S1. PrimeScript™ RT reagent Kit (TaKaRa, JAP) was used for reverse transcription. In total, 1 μl of RT product diluted with 20 μl of ddH₂O was used as template. Then, qPCR was performed in 15 μl of reaction mixture containing 7.5 μl of 2× SYBR^® Premix Ex Taq™ II (TaKaRa, JAP), 1.5 μl of cDNA template, and 0.3 μl of each gene-specific primer. In total, we performed two biological replicates and three technical replicates using the LightCycler^® 480 II RT-PCR System (Roche, SWIT) and its relative quantification software. The parameters of reactions were: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The cDNA libraries were standardized to reference gene 18S. The 2^−ΔΔCt method was used for evaluating gene expression.