Abstract
BackgroundConsidering 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.ResultsHere 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.ConclusionAltogether, 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.
Highlights
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
The Expression Levels of Candidate Housekeeping Genes We chose to investigate seven housekeeping genes commonly used as internal controls in expression studies, ACTB, glyceraldeyde-3-phosphate dehydrogenase (GAPDH), GUSB, HMBS, HPRT1, TBP and 18S rRNA (Table 1)
According to articles published over the past two years, three of them, ACTB, GAPDH and 18S rRNA, collectively correspond to the endogenous controls applied in more than 80% of expression analyses performed on glioma tumors or cell lines
Summary
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. Given the extreme sensitivity of qRT-PCR, a careful and stringent selection of a proper constitutively expressed control gene is required to account for differences in the amount and quality of starting RNA and in cDNA synthesis efficiency. Adequate normalizations presume the use of an internal control, often referred to as a housekeeping or reference gene, whose expression levels should not significantly vary among tissues and experimental situations analyzed [1,2]. Several reports have mentioned these classical housekeeping genes as showing variable expression levels in different experimental conditions [3,4,5,6,7,8,9]. It has been strongly suggested that more than one stable expressed reference gene should be used to avoid misinterpretation of gene expression data [6,7,11,12,13]
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