Total RNA was extracted using TRIzol reagent protocol (Life Technologies), and RNA was quantitated by spectrophotometry at 260 nm using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). Then cDNA synthesis was performed using the SuperScript First-Strand Synthesis System for real-time PCR (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Quantitative PCR analyses for LDHA and HK2 were performed using Assay-on-Demand primers and TaqMan Universal PCR Master Mix reagent (TransGen Biotech, China). Samples were analyzed using an FX96™ Real-Time PCR Detection System (Bio-Rad). The ΔΔCT method was used to quantify relative changes in gene expression as described previously. Expression levels of β-actin were used to normalize the relative expression levels. Each sample was tested at least three times. Primers used for PCR were as follows:
The protocol of real-time PCR were as follows: 1) 94°C for10 minutes, 2) 94°C for 1 minute, 3) 60°C for 30 seconds, 4) 72°C for 1 minute, 5) repeat the steps 1–4, 6) 72°C for 10 minutes, and 7) 4°C forever.
The protocol of real-time PCR were as follows: 1) 94°C for10 minutes, 2) 94°C for 1 minute, 3) 60°C for 30 seconds, 4) 72°C for 1 minute, 5) repeat the steps 1–4, 6) 72°C for 10 minutes, and 7) 4°C forever.