Quantitative RT-PCR Methodology

qRT-PCR was carried out as described previously (64 (link), 65 (link)). Total RNA of each sample was isolated with TRIzol (Invitrogen, USA). The RNA was then quantified spectrophotometrically. qRT-PCR was carried out on 1 μg of total RNA by using an EvoM-MLV RT kit with gDNA clean for qPCR (AG11705; Accurate Biotechnology) according to the manufacturer’s instructions. The cDNAs were synthesized with a reverse transcription kit and used as the template for qRT-PCR. qRT-PCR was performed in 384-well plates with a total volume of 10 μl containing 5 μl 2× SYBR green premix pro Taq HS qPCR kit (AG11701; Accurate Biotechnology), 2.6 μl H₂O, 2 μl cDNA template, and 0.2 μl each of forward and reverse primers (10 μM). All samples were tested in biological triplicate and run on a CFX384 Touch (Bio-Rad, USA) according to the manufacturer’s instructions. The cycling parameters were 95°C for 30 s to activate the polymerase; 40 cycles of 95°C for 10 s; and 56°C for 30 s. Fluorescence measurements were performed at 72°C for 1 s during each cycle. Cycling was terminated at 95°C with a calefactive velocity of 5°C/s to obtain a melting curve. The expression of samples was normalized by β-actin, gapdh, and ef1α using the 2^−ΔΔCT method (66 (link)). All the primers are listed in Table S1B.