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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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Main Authors: Neuvians, Tanja Pascale (Author) , Gashaw, Isabella (Author) , Sauer, Christian (Author) , Ostau, Christian Eike von (Author) , Kliesch, Sabine (Author) , Bergmann, Martin (Author) , Häcker, Axel (Author) , Grobholz, Rainer (Author)
Format: Article (Journal)
Language:English
Published: [4 May 2005]
In: Journal of biotechnology
Year: 2005, Volume: 117, Issue: 2, Pages: 163-171
ISSN:1873-4863
DOI:10.1016/j.jbiotec.2005.01.011
Online Access: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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Author Notes:Tanja Pascale Neuvians, Isabella Gashaw, Christian Georg Sauer, Christian von Ostau, Sabine Kliesch, Martin Bergmann, Axel Häcker, Rainer Grobholz
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Summary: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.
Item Description:Gesehen am 08.06.2022
Physical Description:Online Resource
ISSN:1873-4863
DOI:10.1016/j.jbiotec.2005.01.011

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