Real-Time Quantitative PCR

The total RNA of cells was isolated using RNA extraction kit (Omega, USA) and assayed by a NanoDrop system. A 20 μL reaction system was prepared according to the instructions of the reverse transcription reaction kit: 1) 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, 200 ng of total RNA, dd H₂O to a total volume of 10μL, reacting at 42 °C for 2 min. 2) 10 μL of the reaction solution: 1 μL of Prime Script RT Enzyme Mix I, 1 μL of RT Prime Mix and 4 μL of 5 × Prime Script Buffer 2 and 4 μL of ddH₂O. The reaction conditions were 37°C for 15 min → 85°C for 5 s → 4°C. RT-qPCR reaction system: 10 μL of SYBR^® Premix Ex Taq II (2×), 0.8μL of PCR Forward Primer (10 μM), 0.8 μL of PCR Reverse Primer (10 μM) and 0.4 μL of ROX Reference Dye (50×), 200 ng of RT reaction solution (cDNA solution), addition of ddH2O to a total volume of 20μL. Reaction conditions: 95°C for 30 s; followed by 40 cycles at 95°C for 5 s, 60° C for 30 s. After reaction completion, the amplification curve and the dissolution curve were confirmed. The mRNA levels were normalized against the mRNA level of β-actin and are represented as a fold change. The primers used are listed in Table 1 .

The sequences of primers for RT-PCR.

IL-6, Interleukin-6; IL-1β, Interleukin-1β; TNF-α, Tumor necrosis factor-alpha.