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Open Access Highly Accessed Methodology article

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Valeria Valente12*, Silvia A Teixeira1, Luciano Neder3, Oswaldo K Okamoto4, Sueli M Oba-Shinjo5, Suely KN Marie5, Carlos A Scrideli6, Maria L Paçó-Larson2 and Carlos G Carlotti1

Author affiliations

1 Department of Surgery and Anatomy, Faculty of Medicine, University of São Paulo, Av. dos Bandeirantes 3900, 140490-900, Ribeirão Preto, SP, Brazil

2 Department of Cellular and Molecular Biology, Faculty of Medicine, University of São Paulo, Av. dos Bandeirantes 3900, 140490-900, Ribeirão Preto, SP, Brazil

3 Department of Pathology, Faculty of Medicine, University of São Paulo, Av. dos Bandeirantes 3900, 140490-900, Ribeirão Preto, SP, Brazil

4 Department of Neurology and Neurosurgery, Federal University of São Paulo, R. Botucatu 740, 04023-900, São Paulo, SP, Brazil

5 Department of Neurology, School of Medicine, University of São Paulo, Av. Dr. Arnaldo 455, 01246903, São Paulo, SP, Brazil

6 Department of Pediatrics, Faculty of Medicine, University of São Paulo, Av. dos Bandeirantes 3900, 140490-900, Ribeirão Preto, SP, Brazil

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Citation and License

BMC Molecular Biology 2009, 10:17  doi:10.1186/1471-2199-10-17

Published: 3 March 2009

Abstract

Background

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.

Results

Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold.

Conclusion

Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.