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


CONCLUSIONS
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.
Valente
Valeria
V
Department of Surgery and Anatomy, Faculty of Medicine, University of São Paulo, Av, dos Bandeirantes 3900, 140490-900, Ribeirão Preto, SP, Brazil. vvalente@usp.br
Teixeira
Silvia A
SA
Neder
Luciano
L
Okamoto
Oswaldo K
OK
Oba-Shinjo
Sueli M
SM
Marie
Suely K N
SK
Scrideli
Carlos A
CA
Paçó-Larson
Maria L
ML
Carlotti
Carlos G
CG
D001932:Brain Neoplasms / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D020043:Genes, Essential / Q000235:genetics; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics; D008954:Models, Biological; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D035181:TATA-Box Binding Protein / Q000235:genetics
D004268:DNA-Binding Proteins; C524371:HJURP protein, human; D035181:TATA-Box Binding Protein; C509817:TBP protein, human; D007041:Hypoxanthine Phosphoribosyltransferase