Qs3 real time pcr system

Total RNA of the cells (THP-1 and HUVEC) was extracted using a HiGene Total RNA prep kit (BIOFACT Co. Ltd., Daejeon, Korea). Using a One-Step SYBR PrimeScript RT-PCR kit (TaKaRa Bio Inc., Shiga, Japan), RT-qPCR was performed on a QS3 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The PCR amplification was performed for 40 cycles, with denaturation at 95 °C for 5 sec, and annealing and extension at 60 °C for 60 sec. Each sample was performed in triplicates, including no template control, and the results were subsequently analyzed. The differential expression levels were analyzed using the 2–∆∆Ct method and expressed as relative quantity (RQ). The primer sequences for each gene were as follows: human p62 forward, 5′-TGTGTAGCGTCTGCGAGGGAAA-3′; reverse, 5′-AGTGTCCGTGTTTCACCTTCCG-3′; human LAMP-2 forward, 5′-GGCAATGATACTTGTCTGCTGGC-3′; reverse, 5′-GTAGAGCAGTGTGAGAACGGCA-3′; human NEDD4 forward, 5′-CAGAAGAGGCAGCTTACAAGCC-3′; reverse, 5′-CTTCCCAACCTGGTGGTAATCC-3′; human CUL4 forward, 5′-GAATGAGCGGTTCGTCAACCTG-3′; reverse, 5′-CTGTGGCTTCTTTGTTGCCTGC-3′; human β-actin forward 5′-CACCATTGGCAATGAGCGGTTC-3′; and reverse 5′-AGGTCTTTGCGGATGTCCACGT-3′.