Quantitative Real-Time PCR Protocol

RNA was extracted using the RNeasy Mini Kit (Qiagen, Tokyo, Japan), according to manufacturer’s instructions. Reverse transcription of mRNA was performed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), and the cDNA produced was subjected to qPCR amplification in a PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers used are listed in Table S1. Calibration curves were used to determine the amplification of each target gene with respect to the expression of a reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mean crossing threshold (Ct) values for each target were calculated using Sequence Detection System software v2.3 (Thermo Fisher Scientific)^{37 (link)}.