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 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 beta(2)-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 VEGF(121) 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 tissues VEGF(121) and VEGF(165) accounted for 94% (SD = 7.6%) and VEGF(189) 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.
Wellmann
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Department of Pediatric Oncology/Hematology, Charité Medical Center, Campus Virchow-Klinikum, Humboldt-University at Berlin, 13353 Berlin, Germany. sven.wellmann@charite.de
Taube
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Paal
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Graf V  Einsiedel
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Geilen
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Seifert
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Eckert
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Henze
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Seeger
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D017398:Alternative Splicing; D004587:Electrophoresis, Agar Gel; D016228:Endothelial Growth Factors / Q000235:genetics*; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors