Mx3005p rt qpcr system

A cDNA sample (1 µg; ×10 dilution) was used for qPCR with SYBR^® Premix Ex Taq^™ (Takara Biotechnology Co., Ltd., Dalian, China). qPCR amplification was conducted in a 20 µl reaction mixture containing 10 µl SYBR^® Premix Ex Taq^™, 0.5 µl upstream and downstream primers with a concentration 10 µmol/l and 8 µl nuclease-free double-distilled water in the Mx3005P RT-qPCR System (Stratagene; Agilent Technologies Inc., Santa Clara, CA, USA). The thermocycling conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 sec, 60°C for 20 sec, 72°C for 20 sec and 95°C for 1 min, 55°C for 30 sec and 95°C for 30 sec. Following the reaction, 10 µl assay mixture was used for 1.5% agarose gel electrophoresis using Tris, acetic acid and EDTA buffer (1x), following a standard procedure. GAPDH was used as the reference gene and each sample was run at least in triplicate. The results were quantified using the 2^−∆∆Cq method (13 (link)). All PCR primers were designed with Primer Premier 6.0 (VoyaGene Biotech Co., Ltd., Hangzhou, China) according to the mRNA sequence from the National Centre for Biotechnology Information (https://www.ncbi.nlm.nih.gov/; ACSL1, NM_001995; GAPDH, NM_001256799) and synthesized by Shanghai Biological Technology Ltd. (Shanghai, China) and the sequences are presented in Table I.