Development of a multiplexed PCR-coupled liquid bead array assay for vascular endothelial growth factor (VEGF) splice variants

ObjectivesTo develop a multiplex PCR-coupled liquid bead array assay for the expression of VEGF splice variants.Design and methodsThe assay was based on the combination of multiplex PCR with liquid bead array technology, and optimized and evaluated in terms of analytical sensitivity, specificity, and reproducibility using the MCF-7 cell line. Clinical performance was evaluated in 16 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissues from NSCLC patients.ResultsThe assay is highly sensitive, reproducible and can detect specifically VEGF splice variants in clinical samples. When applied in 32 clinical samples it gave comparable results to RT-qPCR (concordance of 81%, 75%, 88% and 81% for PBGD, VEGF121, VEGF165, and VEGF189 respectively).ConclusionsThe developed assay can simultaneously detect three VEGF splice variants with high specificity and sensitivity and can be further used to evaluate the role of each individual VEGF splice variant in molecular therapies targeted against VEGF.