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Validation of Reference Genes for Expression Studies in Human Meningiomas under Different Experimental Settings

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Abstract

Quantitative polymerase chain reaction (qPCR) is a sensitive technique for the quantitative analysis of gene expression levels. To compare mRNA transcripts across tumour and non-pathological tissue, appropriate reference genes are required for internal standardisation. Validation of these reference genes in meningiomas has not yet been reported. After mRNA transcription of meningioma (WHO grade I-III) and meningeal tissue from three different experimental sample types (fresh tissue, primary cell cultures and FFPE tissue), 13 candidate reference genes (ACTB, B2M, HPRT, VIM, GAPDH, YWHAZ, EIF4A2, MUC1, ATP5B, GNB2L, TUBB, CYC1, RPL13A) were chosen for quantitative expression analysis. Two statistical algorithms (GeNorm and NormFinder) were used for validation of gene expression stability. All candidate housekeepers tested for stability were checked within and across the three tissue analysis groups. Pearson correlation, the ΔC t method and ranking analysis identified the most non-regulated genes suitable for internal standardisation. TUBB, HPRT and ACTB were the most stably expressed genes for all analysis groups across meningioma and non-pathological meningeal tissue combined. In contrast, analysis of the consistency of reference gene expression within specific meningioma and meningeal tissues resulted in specific reference gene rankings for each tissue type. Future gene expression analyses require reference genes to be chosen that are suitable for the tissue types and for the experimental paradigms being studied. Validation of candidate housekeeper genes in meningiomas for quantitative real-time polymerase chain reaction revealed for the first time TUBB, ACTB and HPRT as the most consistently expressed genes among meningioma and non-pathological meningeal tissue across a range of experimental settings.

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Author notes

  1. Diana Freitag and Arend Koch both authors contributed equally to this work

Authors and Affiliations

  1. Department of Neurosurgery, Section of Experimental Neurooncology, Jena University Hospital – Friedrich Schiller University Jena, Am Klinikum 1, 07747, Jena, Germany

    Diana Freitag, Aaron Lawson McLean, Rolf Kalff & Jan Walter

  2. Institute of Neuropathology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 - Virchowweg 15, 10117, Berlin, Germany

    Arend Koch

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  1. Diana Freitag
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Correspondence to Diana Freitag.

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Freitag, D., Koch, A., Lawson McLean, A. et al. Validation of Reference Genes for Expression Studies in Human Meningiomas under Different Experimental Settings. Mol Neurobiol 55, 5787–5797 (2018). https://doi.org/10.1007/s12035-017-0800-3

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  • DOI: https://doi.org/10.1007/s12035-017-0800-3

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