Quantification of Angiogenic Factors in RAW 264.7 Cells

After stimulation with TNF-α (10 ng/ml), RNA was extracted from treated RAW 264.7 cells by using Trizol reagent (Invitrogen). cDNA synthesis involved use of the PrimeScript RT reagent Kit with gDNA Eraser (TakaRa Biotechnology, Dalian, China). The mRNA expression of VEGF-A, ANGPT-1 and ANGPT-2 was quantified by qRT-PCR with SYBR Green qPCR Master Mix reagent (Takara Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences of forward and reverse primers for VEGF-A, ANGPT-1, ANGPT-2 and GAPDH are in Table 2. The 2^−ΔΔCT method was used to analyze relative expression between treatments [40 (link)]. The results were normalized against GAPDH expression.