Quantifying Endothelial Cell Gene Expression

The expression of 84 genes related to endothelial cell biology (catalogue number PAHS-015ZD-24, Qiagen, Hilden, Germany) by HUVEC was determined using QuantiTect Primer Assay (Qiagen, Hilden, Germany) in a real-time polymerase chain reaction (RT-PCR) as previously published [2 (link)]. Briefly, total RNA from HUVEC pretreated with DBMSCs and 100 μM H₂O₂ for 48 h was isolated, and cDNA was then synthesized using FastLane Cell cDNA kit and RT Primer Mix (Qiagen) as previously published [2 (link)]. After quantifying mRNA using QuantiTect SYBR Green PCR Kit (Qiagen), the real-time PCR reaction was performed in triplicate on the CFX96 real-time PCR detection system (BIO-RAD) as previously published [2 (link)]. To analyze the data, the CFX manager software (Bio-Rad, CA, USA) was used. The results were exported to Microsoft Excel for further analysis. The results were expressed as fold change by calculating the ΔΔ⁻² values. The relative expression of an internal house-keeping gene as a loading control was used as provided in the kit. Experiments were performed in triplicate using HUVEC prepared from independent umbilical cord tissue and repeated three times.