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Clinical Chemistry 47: 654-660, 2001;
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(Clinical Chemistry. 2001;47:654-660.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Specific Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor (VEGF) Splice Variants by LightCycler Technology

Sven Wellmanna,1, Tillmann Taube1, Krisztina Paal1, Hagen Graf v. Einsiedel1, Wilhelm Geilen1, Georg Seifert1, Cornelia Eckert1, Günter Henze1 and Karlheinz Seeger1

1 Department of Pediatric Oncology/Hematology, Charité Medical Center, Campus Virchow-Klinikum, Humboldt-University at Berlin, 13353 Berlin, Germany.
a Address correspondence to this author at: Otto-Heubner-Centrum für Kinder- und Jugendmedizin der Charité, Campus Virchow-Klinikum, Forschungshaus R 2.0412, Augustenburger Platz 1, 13353 Berlin, Germany. Fax 49-30-450-59911; e-mail sven.wellmann{at}charite.de.

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 ß2-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.




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