title: Standardization strategy for quantitative PCR in human seminoma and normal testis.
abstract: Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.
authors.affiliation: Department of Pathology, University Hospital Mannheim, Ruprecht-Karls-University Heidelberg, Mannheim, Germany. tanja.neuvians@path.ma.uni-heidelberg.de
authors.forename: Tanja Pascale
authors.initials: TP
authors.lastname: Neuvians
authors.forename: Isabella
authors.initials: I
authors.lastname: Gashaw
authors.forename: Christian Georg
authors.initials: CG
authors.lastname: Sauer
authors.forename: Christian
authors.initials: C
authors.lastname: von Ostau
authors.forename: Sabine
authors.initials: S
authors.lastname: Kliesch
authors.forename: Martin
authors.initials: M
authors.lastname: Bergmann
authors.forename: Axel
authors.initials: A
authors.lastname: Häcker
authors.forename: Rainer
authors.initials: R
authors.lastname: Grobholz
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002138:Calibration; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D018239:Seminoma / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013736:Testicular Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins