Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology

Background: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors , and hematologic malignancies. The expression of isoforms of: VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse: transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formatio n. Methods: We used a real-time RT-PCR assay on the LightCycler system. Applic ability for detection of different VEGF mRNAs and total VEGF message was te sted on seven healthy tissues (each pooled from healthy donors) and seven c orrelated malignant tissues. Results were normalized to beta (2)-microglobu lin mRNA. Amplification of VEGF splice variants was performed exclusively w ith variant-specific reverse primers, whereas forward primer and fluorescen t probe were common to obtain similar RT-PCR kinetics. Results: Highly specific detection of VEGF splice variants was achieved wit h minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA:e ncoding diffusible variants VEGF,,, and VEGF(165) constituted on average 78 % (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF (189) constituted on average 22% (SD = 5.4%). In contrast, in:malignant tis sues VEGF(121) and VEGF(165) accounted for 94% (SD = 7.6%) and VEGF(189) on ly 6% (SD = 3.7%). Conclusions: Because of the ability for quantification of VEGF splice varia nts with high specificity, sensitivity, and reproducibility, this new Light Cycler assay is superior to conventional semiquantitative competitive RT-PC R and immunological assays and may contribute to better understanding of VE GF-mediated angiogenesis. <(c)> 2001 American Association for Clinical Chem istry.