gms | German Medical Science

57. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e. V. (DGNC)
Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

11. bis 14.05.2006, Essen

Semiquantitative two-step RT real-time PCR for validation of microarray results – Potentials and pitfalls

Semiquantitative Real Time PCR als Validierung für Microarray-Ergebnisse – Potentiale und Probleme

Meeting Abstract

  • corresponding author O. Bozinov - Klinik für Neurochirurgie, Philipps-Universität Marburg
  • S. Köhler - Klinik für Neurochirurgie, Philipps-Universität Marburg
  • L. Benes - Klinik für Neurochirurgie, Philipps-Universität Marburg
  • H. Bertalanffy - Klinik für Neurochirurgie, Philipps-Universität Marburg
  • U. Sure - Klinik für Neurochirurgie, Philipps-Universität Marburg

Deutsche Gesellschaft für Neurochirurgie. Japanische Gesellschaft für Neurochirurgie. 57. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e.V. (DGNC), Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie. Essen, 11.-14.05.2006. Düsseldorf, Köln: German Medical Science; 2006. DocP 06.82

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Veröffentlicht: 8. Mai 2006

© 2006 Bozinov et al.
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Objective: Microarrays have been widely used for the analysis of gene expression, but the issue of reproducibility has yet to be fully resolved. In previous microarray analysis our group detected altered expression levels of the genes DBCCR1, OLIG2, Rab34, WIF-1 and IL13RA2 in astrocytomas of WHO-grade III and IV. Differentially expressed genes were selected by a fold change difference of at least 2 and an absolute value of the t-statistic of 1.96. Despite statistical accuracy genetic results are sometimes not equivalent in confirming tests. To validate our alterations we performed semiquantitative two-step Real-Time PCR examinations for all selected tumour tissues. In addition we analysed also our results for previously presented microarray case reports.

Methods: To study discovered differences in the expression of the five oncogenic genes, we conducted semiquantitative two-step RT PCR examinations on all 17 tissue samples of the large group and additional 4 tissue samples of two progressive glioma case reports. All tissue samples derived from the same specimens of the previously reported microarray studies. Analysis were performed using the ABI™ Sequence Detection System ABI PRISM® 7700 and Qiagen™ QuantiTect® SYBR® Green PCR Kits.

Results: Results from array analysis of IL13RA2 (down-regulation) and OLIG2 (up-regulation) for the large group were confirmed by RT PCR for the same direction of expression. However, Rab34 showed significant down-regulation on array studies (folde change -2,22) and opposite up-regulation (+2,07) on RT PCR testing. DBCCR1, WIF-1 and IL13RA2 were confirmed with the right expression direction for 2 case reports with progressive malignant glioma tissues.

Conclusions: Our RT PCR results in the same specimen group confirm previous microarray results for most genes. But particulary completely for two previous genetic case reports with extensive array analysis. On the other hand, Rab34 was not confirmed for the large group of malignant gliomas, instead it showed an opposite expression profile of this Ras-like GTP binding protein, which is involved in various steps along the exocytic and endocytic pathways. Despite statistical significance microarray analysis can not be used as validated prediction of expression. RT PCR is still needed and might reveal differences in expected gene expression. However, microarray analysis with a close binary question still has the potential for further successful specific genetic analysis.