The expression level of target messenger RNA (mRNA) was analyzed by real-time quantitative PCR. TRIZOL reagent (Sigma-Aldrich) and RNeasy Mini Kit (Sigma-Aldrich) were used to extract total RNA from samples. Complementary DNA (cDNA) was synthesized with Omniscript reverse transcription (RT) kit (QIAGEN, Hilden, Germany) and qPCR was amplified with SYBR Green (QIAGEN). The CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA) was used for quantitative analysis of RT-qPCR gene expression. Each real-time PCR sample (10 µL) contained 1 µL cDNA and 5 µL SYBR Green (QIAGEN), and the final concentration of forward and reverse primers was 10 µM.
All reactions were carried out at 95 °C for 10 minutes, then denatured at 95 °C for 2 seconds, annealed at 60 °C for 20 seconds, and extended at 70 °C for 10 seconds. Forty cycles were performed. A melt curve was produced as temperature was increased from 70 °C to 95 °C in 0.5 °C increments within 5 seconds to ensure specificity of amplification. Taking GAPDH as reference, the relative expression level was calculated using the ΔΔCt method. The primer sequences are shown inTable 1 .
All reactions were carried out at 95 °C for 10 minutes, then denatured at 95 °C for 2 seconds, annealed at 60 °C for 20 seconds, and extended at 70 °C for 10 seconds. Forty cycles were performed. A melt curve was produced as temperature was increased from 70 °C to 95 °C in 0.5 °C increments within 5 seconds to ensure specificity of amplification. Taking GAPDH as reference, the relative expression level was calculated using the ΔΔCt method. The primer sequences are shown in