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67th Annual Meeting of the German Society of Neurosurgery (DGNC)
Joint Meeting with the Korean Neurosurgical Society (KNS)

German Society of Neurosurgery (DGNC)

12 - 15 June 2016, Frankfurt am Main

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Validation of appropriate reference genes for transcription analysis of human meningiomas

Meeting Abstract

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  • Diana Freitag - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany
  • Fanny Rapp - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany
  • Susanne Grube - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany
  • Rolf Kalff - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany
  • Christian Ewald - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany
  • Jan Walter - Department of Neurosurgery, University Hospital Jena, Friedrich-Schiller-University, Jena, Germany

Deutsche Gesellschaft für Neurochirurgie. 67. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), 1. Joint Meeting mit der Koreanischen Gesellschaft für Neurochirurgie (KNS). Frankfurt am Main, 12.-15.06.2016. Düsseldorf: German Medical Science GMS Publishing House; 2016. DocP 045

doi: 10.3205/16dgnc420, urn:nbn:de:0183-16dgnc4205

Published: June 8, 2016

© 2016 Freitag et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.

Outline

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Objective: In meningiomas transcription analysis performed by qRT-PCR is used as a sensitive technique for quantitative analysis of gene expression levels. To compare mRNA-transcripts across different tumor samples, one or various appropriate reference genes are required for means of internal standardization. Validation of those reference genes in meningiomas has not been reported yet.

Method: After mRNA-transcription of meningioma (WHO I°-III°) and dural tissue samples, 12 candidate reference genes: B2M, HPRT, Vimentin, GAPDH, YWHAZ, EIF4A2, MUC1, ATP5B, GNB2L, TUBB, CYC1, RPL13A were chosen for quantitative expression analysis. GeNorm and Normfinder, two established statistical algorithms, were used for validation of gene expression stability. For all tested candidate genes we measured stability within and among the four tissue groups. Pearson correlation and ranking analysis identified the most non-regulated genes for internal standardization.

Results: After hierarchical evaluation of the 3 analysis methods by ranking analysis, RPL13A (MS=1.33), GAPDH (MS=3.33), GNB2L (MS=3.67) and CYC1 (MS=4.00) were found the most constantly expressed genes among all meningioma grades and non-pathological dural tissue. Combination of GAPDH+CYC1 expression appeared to be even more suitable for normalization of specific target genes. But in contrast, analysis of the consistency of reference gene expression within the particular WHO grades delivered various ratios, depending on the respective tissue to be analyzed.

Conclusions: Validation of candidate reference genes in meningiomas for quantitative real-time polymerase chain reaction revealed for the first time RPL13A, GAPDH, GNB2L and CYC1 as well as the combination GAPDH+CYC1 as the most constantly expressed genes among all meningioma grades. For further meningioma gene expression studies it is mandatory to use only validated reference genes or combinations and not to rely on typical housekeeper genes, known from other tissue samples.