Quantitative Real-Time PCR Protocol

Total RNA was extracted using the RNA extraction kit from Axgen (Union City, CA, United States) according to the manufacturer’s instructions. After extraction, the RNA samples were treated with gDNase to remove DNA, then reverse-transcribed (1 μg per sample) to cDNA using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Quantitative Real-Time PCR was conducted using SYBR Premix ExTaqII (2×) Kit (Takara, Tokyo, Japan) on an iCycler Iq TM Multicolor PCR Detection System (Bio-Rad, Hercules, CA, United States; Li et al., 2017 (link)). β-ACTIN was used as an internal control gene. Primers used for gene expression analyses are listed in Supplementary Table S1. The relative expression of genes was calculated using the 2^−ΔΔCT method as reported in Livak and Schmittgen (2001) (link).