Quantitative PCR for gene expression in endothelial cells.

Endothelial cells were cultured and infected with F. nucleatum in order to elucidate the role of F. nucleatum in modulating the expression of target genes. Total RNA was extracted using TRIzol reagent and was quantified in a spectrophotometer at A₂₆₀. RNA samples with a 260:280 ratio (around 2.0) were employed to ensure high purity. Total RNA (1 μg) from each sample was used for reverse transcription. Each assay was carried out in triplicate using a 20-μl reaction mixture. Experiments were performed at least three times and in triplicate. The primers used for real-time PCR analysis (purchased from Life Technologies) were as follows: Hs02585826_cn (VEGF growth factor A [VEGFA]), Hs04047136_cn (PLCG1), Hs00164893_CE (inducible nitric oxide synthase [iNOS]), and Hs00725652_CE (endothelial NOS [eNOS]). β-actin (Hs01060665_g1) was used as the housekeeping gene control for normalization. Data analysis was performed using the threshold cycle (ΔΔC_T) method, and β-actin was used as the reference gene.