RNA Isolation and Real-Time PCR Analysis

The primers were designed and synthesized by Genscript Biotech Co. Ltd. (Nanjing, China). Total RNA was extracted by using TRIzol reagent (Ambion, Foster City, CA, United States) according to the instructions. Concentration and integrity of total RNA were verified by a NanoDrop 2000 nano-spectrophotometer (Thermo, Waltham, MA, United States). Reverse transcription (RT) was proceeded by using SuperScript^® III First-Strand Synthesis Super Mix for qRT-PCR Kit (Invitrogen, Carlsbad, CA, United States), and real-time PCR reactions were performed using Platinum^® SYBR^® Green qPCR SuperMix-UDG with ROX Kit (Invitrogen, Carlsbad, CA, United States). The reaction systems were prepared following the instructions of the kits and reacted on an Applied Biosystems 7500/7500 Fast Real-Time PCR System (AB, Foster City, CA, United States). The initial enzyme activation step was at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s. At the end of PCR, to evaluate specific amplification of the target genes, melting curve ranging from 60 to 95°C were also included in each run.