Quantitative Real-Time RT-PCR for Gene Expression Analysis

Quantitative real-time RT-PCR was carried out as previously described with minor modifications (14 (link)). Briefly, total RNA of 1 µg was reversely transcribed in a 20 μl of reaction using Evo M-MLV RT Premix for qPCR (AG, Changsha, China, code no: AG11706) according to the manufacturer’s protocol. The reaction products were then diluted with 40 μl of distilled water. The real-time PCR reaction was used of 2 μl of diluted reverse transcription product, 10 µl of 2× SYBR^® Green Pro Taq HS Premix (SYBR^® Green Premix Pro Taq HS qPCR Kit, code no: AG11606), and 0.6 µl of forward and reverse primers (0.3 μM). The reaction was performed in a Light Cycler@ 480 II Sequence Detection System (Roche, Basel, Switzerland) for 45 cycles (95°C for 30 s and 60°C for 5 s) after an initial 30-s denaturation at 95°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The RNA levels of tumor samples and paired adjacent samples were calculated using the 2^−ΔCt method. All primers sequences of the hub genes and GAPDH were listed in Supplementary Table 1.