Quantitative RT-PCR Gene Expression Analysis

RT-qPCR was performed using the same batch of specimens. The RNAs were extracted using TRIzol reagent, reverse transcribed using Moloney murine leukemia virus (Takara Biotechnology Co., Ltd., Shiga, Japan), and separately subjected to PCR amplification. The primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) using GAPDH as the reference gene. The primer sequences, amplification length and annealing temperature are reported previously [10 (link)]. The reaction procedure was as follows: Initial denaturation at 94°C for 5 min; followed by 35 cycles for denaturation at 94°C for 30 sec, annealing for 30 sec and elongation at 72°C for 30 sec; each sample was assayed in triplicate. A temperature range of 65–95°C was selected for drawing melting curves.