DNA microarray

Total RNA was isolated using an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Genomics DNA was removed with DNase I (QIAGEN, Hilden, Germany). Synthesis of target cRNA was performed using Agilent’s Low RNA Input Linear Amplification Kit PLUS (Agilent Technology, CA, USA) according to the manufacturer’s instructions. Briefly, each 0.2 μg of total RNA was mixed with the diluted Spike mix and T7 promoter primer mix, then incubated at 65°C for 10 min. Next, cDNA master mix (5× First strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mixer. The procedures for DNA microarray were identical to those described in our previous study (Wang et al., 2014). All data normalization and selection of fold-changed genes was performed using GeneSpring GX 7.3 (Agilent Technology). Genes were filtered by removing flag-out genes in each experiment. The averages of normalized ratios were calculated by dividing the test channel intensity by the control channel intensity.