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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| Veröffentlicht in: | Journal of biotechnology 2005-05, Vol.117 (2), p.163-171 |
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| creator | Neuvians, Tanja Pascale Gashaw, Isabella Sauer, Christian Georg Ostau, Christian von Kliesch, Sabine Bergmann, Martin Häcker, Axel Grobholz, Rainer |
| description | 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. |
| doi_str_mv | 10.1016/j.jbiotec.2005.01.011 |
| format | Article |
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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.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2005.01.011</identifier><identifier>PMID: 15823405</identifier><identifier>CODEN: JBITD4</identifier><language>eng</language><publisher>Lausanne: Elsevier B.V</publisher><subject>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</subject><ispartof>Journal of biotechnology, 2005-05, Vol.117 (2), p.163-171</ispartof><rights>2005 Elsevier B.V.</rights><rights>2005 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c424t-e0f85cd51c11e9fec76538d03947fac60f53c5e3b292ca12117290226d1ac66c3</citedby><cites>FETCH-LOGICAL-c424t-e0f85cd51c11e9fec76538d03947fac60f53c5e3b292ca12117290226d1ac66c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jbiotec.2005.01.011$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,27903,27904,45974</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16704920$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15823405$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Neuvians, Tanja Pascale</creatorcontrib><creatorcontrib>Gashaw, Isabella</creatorcontrib><creatorcontrib>Sauer, Christian Georg</creatorcontrib><creatorcontrib>Ostau, Christian von</creatorcontrib><creatorcontrib>Kliesch, Sabine</creatorcontrib><creatorcontrib>Bergmann, Martin</creatorcontrib><creatorcontrib>Häcker, Axel</creatorcontrib><creatorcontrib>Grobholz, Rainer</creatorcontrib><title>Standardization strategy for quantitative PCR in human seminoma and normal testis</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>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.</description><subject>Beta-actin</subject><subject>Biological and medical sciences</subject><subject>Biomarkers, Tumor - genetics</subject><subject>Biomarkers, Tumor - metabolism</subject><subject>Biotechnology</subject><subject>Calibration</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>GAPDH</subject><subject>Gene Expression Profiling - methods</subject><subject>Gene Expression Profiling - standards</subject><subject>Genetic Testing - methods</subject><subject>Genetic Testing - standards</subject><subject>Housekeeping gene</subject><subject>Humans</subject><subject>Male</subject><subject>Neoplasm Proteins - genetics</subject><subject>Neoplasm Proteins - metabolism</subject><subject>Oligonucleotide Array Sequence Analysis - methods</subject><subject>Oligonucleotide Array Sequence Analysis - standards</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Polymerase Chain Reaction - standards</subject><subject>Quantitative PCR</subject><subject>Reference Standards</subject><subject>Reproducibility of Results</subject><subject>Seminoma</subject><subject>Seminoma - genetics</subject><subject>Seminoma - metabolism</subject><subject>Sensitivity and Specificity</subject><subject>Testicular Neoplasms - diagnosis</subject><subject>Testicular Neoplasms - genetics</subject><subject>Testicular Neoplasms - metabolism</subject><subject>Testis</subject><subject>Testis - metabolism</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFLHTEQx4NU9Gn7EZRc2tu-ZrKbZPck8mhtQaja9hzysrOax26iSVawn76Rt-BRGJjD_P4z4RdCzoCtgYH8ulvvti5ktGvOmFgzKAUHZAWtqqumlfUHsipcW4EU8picpLRjjDWdgCNyDKLldcPEitz-zsb3Jvbun8kueJpyNBnvX-gQIn2ajc8ul8kz0pvNHXWePsyTKRhOzofJ0JKmPsTJjDRjyi59JIeDGRN-Wvop-fv925_Nj-r619XPzeV1ZRve5ArZ0ArbC7AA2A1olRR127O6a9RgrGSDqK3Aess7bg1wAMU7xrnsoUylrU_Jl_3exxie5nJaTy5ZHEfjMcxJS6V4qzh_FwTVNkIxVUCxB20MKUUc9GN0k4kvGph-la53epGuX6VrBqWg5M6XA_N2wv4ttVguwOcFMMmacYjGW5feOKnKx3BWuIs9h8Xbs8Ook3XoLfYuos26D-6dp_wHqy-idw</recordid><startdate>20050504</startdate><enddate>20050504</enddate><creator>Neuvians, Tanja Pascale</creator><creator>Gashaw, Isabella</creator><creator>Sauer, Christian Georg</creator><creator>Ostau, Christian von</creator><creator>Kliesch, Sabine</creator><creator>Bergmann, Martin</creator><creator>Häcker, Axel</creator><creator>Grobholz, Rainer</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20050504</creationdate><title>Standardization strategy for quantitative PCR in human seminoma and normal testis</title><author>Neuvians, Tanja Pascale ; Gashaw, Isabella ; Sauer, Christian Georg ; Ostau, Christian von ; Kliesch, Sabine ; Bergmann, Martin ; Häcker, Axel ; Grobholz, Rainer</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c424t-e0f85cd51c11e9fec76538d03947fac60f53c5e3b292ca12117290226d1ac66c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Beta-actin</topic><topic>Biological and medical sciences</topic><topic>Biomarkers, Tumor - genetics</topic><topic>Biomarkers, Tumor - metabolism</topic><topic>Biotechnology</topic><topic>Calibration</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>GAPDH</topic><topic>Gene Expression Profiling - methods</topic><topic>Gene Expression Profiling - standards</topic><topic>Genetic Testing - methods</topic><topic>Genetic Testing - standards</topic><topic>Housekeeping gene</topic><topic>Humans</topic><topic>Male</topic><topic>Neoplasm Proteins - genetics</topic><topic>Neoplasm Proteins - metabolism</topic><topic>Oligonucleotide Array Sequence Analysis - methods</topic><topic>Oligonucleotide Array Sequence Analysis - standards</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Polymerase Chain Reaction - standards</topic><topic>Quantitative PCR</topic><topic>Reference Standards</topic><topic>Reproducibility of Results</topic><topic>Seminoma</topic><topic>Seminoma - genetics</topic><topic>Seminoma - metabolism</topic><topic>Sensitivity and Specificity</topic><topic>Testicular Neoplasms - diagnosis</topic><topic>Testicular Neoplasms - genetics</topic><topic>Testicular Neoplasms - metabolism</topic><topic>Testis</topic><topic>Testis - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Neuvians, Tanja Pascale</creatorcontrib><creatorcontrib>Gashaw, Isabella</creatorcontrib><creatorcontrib>Sauer, Christian Georg</creatorcontrib><creatorcontrib>Ostau, Christian von</creatorcontrib><creatorcontrib>Kliesch, Sabine</creatorcontrib><creatorcontrib>Bergmann, Martin</creatorcontrib><creatorcontrib>Häcker, Axel</creatorcontrib><creatorcontrib>Grobholz, Rainer</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Neuvians, Tanja Pascale</au><au>Gashaw, Isabella</au><au>Sauer, Christian Georg</au><au>Ostau, Christian von</au><au>Kliesch, Sabine</au><au>Bergmann, Martin</au><au>Häcker, Axel</au><au>Grobholz, Rainer</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Standardization strategy for quantitative PCR in human seminoma and normal testis</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2005-05-04</date><risdate>2005</risdate><volume>117</volume><issue>2</issue><spage>163</spage><epage>171</epage><pages>163-171</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><coden>JBITD4</coden><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.</abstract><cop>Lausanne</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>15823405</pmid><doi>10.1016/j.jbiotec.2005.01.011</doi><tpages>9</tpages></addata></record> |
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| 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 |
| title | Standardization strategy for quantitative PCR in human seminoma and normal testis |
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