Total RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis

Total RNA extraction, cDNA synthesis and qRT-PCRs were performed according to Hu et al. (2016) [33 (link)] described. Briefly, total RNA was extracted from fresh tissues using Trizol reagent (Invitrogen, Carlsbad, CA). The first strand cDNA fragments were synthesized from 2 μg of total RNA using oligo(dT)12-18 primer using cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada) after RNA quality and integrity was checked by Nanodrop 2000 and 0.8% agarose gel. Gene-specific primers (Table 2) were designed based on the target gene sequences using Primer 5 software. The qRT-PCRs were performed with ABI7500 in a final volume of 20 μL, with each containing 2 μl of cDNA, 10 μL of 2×SYBR Green qPCR Mix (Takara, Otsu, Shiga, Japan) and 2 μM of the forward and reverse primers. Three independent biological replicates of each sample and two technical replicates of each biological replicate were used for real-time PCR analysis. The thermal cycling conditions were as follows: 40 cycles of 95°C denaturation for 5 s, and 52~55°C annealing and extension for 20 s. To determine relative fold differences for each sample, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the DDCT method as described by Livak and Schmittgen (2001) [40 (link)].