Standardization strategy for quantitative PCR in human seminoma and normal testis.
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.
Neuvians
Tanja Pascale
TP
Department of Pathology, University Hospital Mannheim, Ruprecht-Karls-University Heidelberg, Mannheim, Germany. tanja.neuvians@path.ma.uni-heidelberg.de
Gashaw
Isabella
I
Sauer
Christian Georg
CG
von Ostau
Christian
C
Kliesch
Sabine
S
Bergmann
Martin
M
Häcker
Axel
A
Grobholz
Rainer
R
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*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins