Article
Heterogeneous expression of “reference” genes for expression studies in meningiomas using quantitative RT-PCR
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Authors
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Matthias Seibl-Leven
- Labor für Neuroonkologie und Experimentelle Neurochirurgie, Klinik für Allgemeine Neurochirurgie, Zentrum für Neurochirurgie, Universitätsklinikum Köln, Köln
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Gabriele Röhn
- Labor für Neuroonkologie und Experimentelle Neurochirurgie, Klinik für Allgemeine Neurochirurgie, Zentrum für Neurochirurgie, Universitätsklinikum Köln, Köln
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Arend Koch
- Institut für Neuropathologie, Campus Charité Mitte, Charté – Universitätsmedizin Berlin, Berlin
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Roland Goldbrunner
- Labor für Neuroonkologie und Experimentelle Neurochirurgie, Klinik für Allgemeine Neurochirurgie, Zentrum für Neurochirurgie, Universitätsklinikum Köln, Köln
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Marco Timmer
- Labor für Neuroonkologie und Experimentelle Neurochirurgie, Klinik für Allgemeine Neurochirurgie, Zentrum für Neurochirurgie, Universitätsklinikum Köln, Köln
| Published: | May 13, 2014 |
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Outline
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Objective: Real-time RT-PCR (RT-qPCR) is often used to analyse gene expression. In human meningiomas, many different reference genes are frequently used, however, no study has been performed yet to validate these “reference genes” with respect to the degree of malignancy. Moreover, no systematic analysis of suitable endogenous control genes exists for primary versus recurrent tumors. Therefore, this analysis was performed in order to enable quantitative gene expression studies in meningiomas including primary and recurrent tumors of WHO°I, °II and °III. The hypothesis was that the reference genes used in other studies remain stable under various conditions.
Method: This study aimed to test five reference genes out of earlier studies (GAPDH, ACTB, SDHA, EIF2B, and MRPL19) for their expression stability in the following groups: peritumoral tissue as control, meningioma WHO °I, recurrent meningioma WHO °I, meningioma WHO °II, recurrent meningioma WHO °II, meningioma WHO °III, and recurrent meningioma WHO °III. Tissue samples (n = 10 per group) were obtained from our tumour tissue bank. After RNA extraction and transcription into cDNA, all qPCRs were done twice in triplicate. The statistical softwares geNorm, BestKeeper and NormFinder were used to select the most stable candidate genes, whereby both the intra- and intergroup variation of candidate genes were modeled.
Results: The CT-values of the reference genes analyzed showed different transcription levels in all groups. Interestingly, all tested candidate genes were differentially expressed between the different groups. Especially between grade II and grade III meningiomas, most tested genes increased their expression. No single gene performed as adequate references gene for meningiomas. Our results indicate that the combination of ACTB and MRPL19 displayed the most stable values for primary and recurrent meningiomas with rising tumor grade (WHO grade l to III). ACTB and MRPL19 show the most stable results as genes which are expressed in brain tumor tissue.
Conclusions: Taken together, our data show the relevance of previous validation of candidate control genes for each experimental design. Interestingly, housekeeping genes in meningiomas seem to be much more heterogeneous compared to gliomas. Therefore, at least a combination of two reference genes is necessary when comparing gene expression in meningiomas.
Note: Matthias Seibl-Leven and Gabriele Röhn contributed equally.
