Specific Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor (VEGF) Splice Variants by LightCycler Technology
S Wellmann, T Taube, K Paal… - Clinical …, 2001 - academic.oup.com
S Wellmann, T Taube, K Paal, H Graf v. Einsiedel, W Geilen, G Seifert, C Eckert, G Henze…
Clinical Chemistry, 2001•academic.oup.comBackground: 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
formation. Methods: We used a real-time RT-PCR assay on the LightCycler system …
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
formation. Methods: We used a real-time RT-PCR assay on the LightCycler system …
Abstract
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 formation.
Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to β2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.
Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).
Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.
Oxford University Press