Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?
► UVA modulates expression of genes involved in dermal re-modelling. ► Accurate gene expression evaluation requires suitable reference genes. ► Human dermal fibroblasts (HDF) cultured under 21% O 2 and under physiological 5% O 2. ► Most suitable reference genes identified for qPCR analysis on HDF, U...
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| Veröffentlicht in: | Journal of biotechnology 2011-12, Vol.156 (3), p.153-162 |
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| creator | Brugè, F. Venditti, E. Tiano, L. Littarru, G.P. Damiani, E. |
| description | ► UVA modulates expression of genes involved in dermal re-modelling. ► Accurate gene expression evaluation requires suitable reference genes. ► Human dermal fibroblasts (HDF) cultured under 21% O
2 and under physiological 5% O
2. ► Most suitable reference genes identified for qPCR analysis on HDF, UVA-treated. ► β-Actin is unsuitable as reference gene for UVA-treated HDF.
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18
J/cm
2 UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF. |
| doi_str_mv | 10.1016/j.jbiotec.2011.09.018 |
| format | Article |
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2 and under physiological 5% O
2. ► Most suitable reference genes identified for qPCR analysis on HDF, UVA-treated. ► β-Actin is unsuitable as reference gene for UVA-treated HDF.
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18
J/cm
2 UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2011.09.018</identifier><identifier>PMID: 21963587</identifier><identifier>CODEN: JBITD4</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>actin ; Actins - genetics ; Biological and medical sciences ; Biotechnology ; Cell Hypoxia - genetics ; Cell Hypoxia - radiation effects ; Cells, Cultured ; Collagen Type I - genetics ; fibroblasts ; Fibroblasts - metabolism ; Fibroblasts - radiation effects ; Fundamental and applied biological sciences. Psychology ; gene expression ; Gene Expression Profiling - methods ; genes ; Human dermal fibroblasts ; Humans ; hypoxia ; Hypoxia/normoxia ; Matrix Metalloproteinase 1 - genetics ; messenger RNA ; Normalization ; normoxia ; oxygen ; photoaging ; quantitative polymerase chain reaction ; Real-Time Polymerase Chain Reaction ; Reference genes ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; reverse transcription ; RT-qPCR ; Skin - cytology ; Ultraviolet Rays ; UVA</subject><ispartof>Journal of biotechnology, 2011-12, Vol.156 (3), p.153-162</ispartof><rights>2011 Elsevier B.V.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2011 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c450t-2dcf31f897189d47c61657b9191cf6cb91503213d2ba5f30a82ec817ef7d95ce3</citedby><cites>FETCH-LOGICAL-c450t-2dcf31f897189d47c61657b9191cf6cb91503213d2ba5f30a82ec817ef7d95ce3</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.2011.09.018$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24685919$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21963587$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Brugè, F.</creatorcontrib><creatorcontrib>Venditti, E.</creatorcontrib><creatorcontrib>Tiano, L.</creatorcontrib><creatorcontrib>Littarru, G.P.</creatorcontrib><creatorcontrib>Damiani, E.</creatorcontrib><title>Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>► UVA modulates expression of genes involved in dermal re-modelling. ► Accurate gene expression evaluation requires suitable reference genes. ► Human dermal fibroblasts (HDF) cultured under 21% O
2 and under physiological 5% O
2. ► Most suitable reference genes identified for qPCR analysis on HDF, UVA-treated. ► β-Actin is unsuitable as reference gene for UVA-treated HDF.
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18
J/cm
2 UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.</description><subject>actin</subject><subject>Actins - genetics</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cell Hypoxia - genetics</subject><subject>Cell Hypoxia - radiation effects</subject><subject>Cells, Cultured</subject><subject>Collagen Type I - genetics</subject><subject>fibroblasts</subject><subject>Fibroblasts - metabolism</subject><subject>Fibroblasts - radiation effects</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gene expression</subject><subject>Gene Expression Profiling - methods</subject><subject>genes</subject><subject>Human dermal fibroblasts</subject><subject>Humans</subject><subject>hypoxia</subject><subject>Hypoxia/normoxia</subject><subject>Matrix Metalloproteinase 1 - genetics</subject><subject>messenger RNA</subject><subject>Normalization</subject><subject>normoxia</subject><subject>oxygen</subject><subject>photoaging</subject><subject>quantitative polymerase chain reaction</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Reference genes</subject><subject>Reference Standards</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>reverse transcription</subject><subject>RT-qPCR</subject><subject>Skin - cytology</subject><subject>Ultraviolet Rays</subject><subject>UVA</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks1u1DAQxyMEotvCIwC-VJyy2HGc2FyqasVHpUqgwnK1HHu89SqJt7ZTtX0h7jwIz4T3Azj25LH1m_nPzN9F8YrgOcGkebeerzvnE-h5hQmZYzHHhD8pZoS3tKx5Q58Ws8zxkjSsOSqOY1xjjGvByPPiqCKioYy3s-LnFVgIMGpAKxgB3areGZWcH5H1Ad18XVyhHI8-DP7OqRKp0aDr-83uoqc-TQHywzSoERkIg-qRdV3wXa9iigjuNj5mIHm0_HH-Hl1E9PtXqXRyI1IoQO9U18OufBZ-gLBT3Vz75NXKjSsU02QcxLMXxTOr-ggvD-dJsfz44fvic3n55dPF4vyy1DXDqayMtpRYLlrChalb3eTx204QQbRtdA4YphWhpuoUsxQrXoHmpAXbGsE00JPi7b7uJvibCWKSg4sa-l6N4KcoBa5omxdJHyW54Jy2LRaZZHtSBx9jACs3wQ0q3EuC5dZMuZYHM-XWTImFzGbmvNcHhakbwPzL-uteBk4PgIpa9TaoUbv4n6sbzvLomXuz56zyUq1CZpbfshLDWbzGjGTibE9A3u2tgyCjdttfYVwAnaTx7pFm_wBaxcwB</recordid><startdate>20111210</startdate><enddate>20111210</enddate><creator>Brugè, F.</creator><creator>Venditti, E.</creator><creator>Tiano, L.</creator><creator>Littarru, G.P.</creator><creator>Damiani, E.</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>FBQ</scope><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>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20111210</creationdate><title>Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?</title><author>Brugè, F. ; Venditti, E. ; Tiano, L. ; Littarru, G.P. ; Damiani, E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c450t-2dcf31f897189d47c61657b9191cf6cb91503213d2ba5f30a82ec817ef7d95ce3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>actin</topic><topic>Actins - genetics</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cell Hypoxia - genetics</topic><topic>Cell Hypoxia - radiation effects</topic><topic>Cells, Cultured</topic><topic>Collagen Type I - genetics</topic><topic>fibroblasts</topic><topic>Fibroblasts - metabolism</topic><topic>Fibroblasts - radiation effects</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gene expression</topic><topic>Gene Expression Profiling - methods</topic><topic>genes</topic><topic>Human dermal fibroblasts</topic><topic>Humans</topic><topic>hypoxia</topic><topic>Hypoxia/normoxia</topic><topic>Matrix Metalloproteinase 1 - genetics</topic><topic>messenger RNA</topic><topic>Normalization</topic><topic>normoxia</topic><topic>oxygen</topic><topic>photoaging</topic><topic>quantitative polymerase chain reaction</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>Reference genes</topic><topic>Reference Standards</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>reverse transcription</topic><topic>RT-qPCR</topic><topic>Skin - cytology</topic><topic>Ultraviolet Rays</topic><topic>UVA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Brugè, F.</creatorcontrib><creatorcontrib>Venditti, E.</creatorcontrib><creatorcontrib>Tiano, L.</creatorcontrib><creatorcontrib>Littarru, G.P.</creatorcontrib><creatorcontrib>Damiani, E.</creatorcontrib><collection>AGRIS</collection><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>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Brugè, F.</au><au>Venditti, E.</au><au>Tiano, L.</au><au>Littarru, G.P.</au><au>Damiani, E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2011-12-10</date><risdate>2011</risdate><volume>156</volume><issue>3</issue><spage>153</spage><epage>162</epage><pages>153-162</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><coden>JBITD4</coden><abstract>► UVA modulates expression of genes involved in dermal re-modelling. ► Accurate gene expression evaluation requires suitable reference genes. ► Human dermal fibroblasts (HDF) cultured under 21% O
2 and under physiological 5% O
2. ► Most suitable reference genes identified for qPCR analysis on HDF, UVA-treated. ► β-Actin is unsuitable as reference gene for UVA-treated HDF.
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18
J/cm
2 UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>21963587</pmid><doi>10.1016/j.jbiotec.2011.09.018</doi><tpages>10</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | actin Actins - genetics Biological and medical sciences Biotechnology Cell Hypoxia - genetics Cell Hypoxia - radiation effects Cells, Cultured Collagen Type I - genetics fibroblasts Fibroblasts - metabolism Fibroblasts - radiation effects Fundamental and applied biological sciences. Psychology gene expression Gene Expression Profiling - methods genes Human dermal fibroblasts Humans hypoxia Hypoxia/normoxia Matrix Metalloproteinase 1 - genetics messenger RNA Normalization normoxia oxygen photoaging quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction Reference genes Reference Standards Reverse Transcriptase Polymerase Chain Reaction reverse transcription RT-qPCR Skin - cytology Ultraviolet Rays UVA |
| title | Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies? |
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