Standardization strategy for quantitative PCR in human seminoma and normal testis

• Published in: Journal of biotechnology Vol. 117; no. 2; pp. 163 - 171
• Main Authors: Neuvians, Tanja Pascale, Gashaw, Isabella, Sauer, Christian Georg, Ostau, Christian von, Kliesch, Sabine, Bergmann, Martin, Häcker, Axel, Grobholz, Rainer
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 04-05-2005; Amsterdam Elsevier; New York, NY
• Subjects: Beta-actin; Biological and medical sciences; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Biotechnology; Calibration; Fundamental and applied biological sciences. Psychology; GAPDH; Gene Expression Profiling - methods; Gene Expression Profiling - standards; Genetic Testing - methods; Genetic Testing - standards; Housekeeping gene; Humans; Male; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide Array Sequence Analysis - standards; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Quantitative PCR; Reference Standards; Reproducibility of Results; Seminoma; Seminoma - genetics; Seminoma - metabolism; Sensitivity and Specificity; Testicular Neoplasms - diagnosis; Testicular Neoplasms - genetics; Testicular Neoplasms - metabolism; Testis; Testis - metabolism; Testis; Seminoma; Beta-actin; Housekeeping gene; GAPDH; Quantitative PCR; Human; Polymerase chain reaction; Actin; Testicle; Quantitative analysis
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2005.01.011

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.