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-t...
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| Hauptverfasser: | , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
[4 May 2005]
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| In: |
Journal of biotechnology
Year: 2005, Jahrgang: 117, Heft: 2, Pages: 163-171 |
| ISSN: | 1873-4863 |
| DOI: | 10.1016/j.jbiotec.2005.01.011 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.jbiotec.2005.01.011 Verlag, lizenzpflichtig, Volltext: https://www.sciencedirect.com/science/article/pii/S0168165605000568 
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| Verfasserangaben: | Tanja Pascale Neuvians, Isabella Gashaw, Christian Georg Sauer, Christian von Ostau, Sabine Kliesch, Martin Bergmann, Axel Häcker, Rainer Grobholz |
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| 245 | 1 | 0 | |a Standardization strategy for quantitative PCR in human seminoma and normal testis |c Tanja Pascale Neuvians, Isabella Gashaw, Christian Georg Sauer, Christian von Ostau, Sabine Kliesch, Martin Bergmann, Axel Häcker, Rainer Grobholz |
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| 520 | |a 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. | ||
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