Determination and Validation of Reference Gene Stability for qPCR Analysis in Polysaccharide Hydrogel-Based 3D Chondrocytes and Mesenchymal Stem Cell Cultural Models
Identifieur interne : 000C10 ( Main/Exploration ); précédent : 000C09; suivant : 000C11Determination and Validation of Reference Gene Stability for qPCR Analysis in Polysaccharide Hydrogel-Based 3D Chondrocytes and Mesenchymal Stem Cell Cultural Models
Auteurs : Wai Hon Chooi [Singapour] ; Ruijie Zhou [Singapour] ; Suan Siong Yeo [Singapour] ; Feng Zhang [Singapour] ; Dong-An Wang [Singapour]Source :
- Molecular Biotechnology [ 1073-6085 ] ; 2013-06-01.
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Abstract
Abstract: Gene expression study is widely used to obtain information of the cell activities and phenotypes. To quantify gene expression, measurement of the mRNA copy number is commonly done by quantitative RT-PCR (RT-qPCR). However, proper reference gene is needed for different tissues to normalize the expression level of different genes accurately. In this study, reference gene determination was done for three-dimensional (3D) artificial tissue constructs in hydrogel. Porcine synovium-derived mesenchymal stem cells (SMSCs) and rabbit chondrocytes were cultured in both alginate and agarose hydrogels to set up four different 3D culture systems to form the artificial tissue constructs. The gene expression levels of candidate genes were determined by RT-qPCR and then analyzed by geNorm, Bestkeeper, and Normfinder. For porcine SMSCs, PPIA, and TBP were selected for tissue in alginate scaffold whereas HPRT and TBP were selected for the agarose scaffold system. On the other hand, HPRT, PPIA, and RPL18 were the stable reference genes for rabbit chondrocytes in alginate scaffold while TBP, RPL5, and RPL18 were selected for rabbit chondrocytes in agarose scaffold. This study has further indicated that suitable reference genes are different for each tissue and study purpose. The reference genes are expressed in different stability when a scaffold of different material is used.
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DOI: 10.1007/s12033-012-9604-x
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York</pubPlace><date type="published" when="2013-06-01">2013-06-01</date><biblScope unit="volume">54</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="623">623</biblScope><biblScope unit="page" to="633">633</biblScope></imprint><idno type="ISSN">1073-6085</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1073-6085</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>3D culture</term><term>Chondrocytes</term><term>Hydrogel</term><term>Mesenchymal stem cells</term><term>Quantitative PCR</term><term>Reference gene</term><term>Tissue engineering</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Gene expression study is widely used to obtain information of the cell activities and phenotypes. To quantify gene expression, measurement of the mRNA copy number is commonly done by quantitative RT-PCR (RT-qPCR). However, proper reference gene is needed for different tissues to normalize the expression level of different genes accurately. In this study, reference gene determination was done for three-dimensional (3D) artificial tissue constructs in hydrogel. Porcine synovium-derived mesenchymal stem cells (SMSCs) and rabbit chondrocytes were cultured in both alginate and agarose hydrogels to set up four different 3D culture systems to form the artificial tissue constructs. The gene expression levels of candidate genes were determined by RT-qPCR and then analyzed by geNorm, Bestkeeper, and Normfinder. For porcine SMSCs, PPIA, and TBP were selected for tissue in alginate scaffold whereas HPRT and TBP were selected for the agarose scaffold system. On the other hand, HPRT, PPIA, and RPL18 were the stable reference genes for rabbit chondrocytes in alginate scaffold while TBP, RPL5, and RPL18 were selected for rabbit chondrocytes in agarose scaffold. This study has further indicated that suitable reference genes are different for each tissue and study purpose. The reference genes are expressed in different stability when a scaffold of different material is used.</div></front></TEI><affiliations><list><country><li>Singapour</li></country></list><tree><country name="Singapour"><noRegion><name sortKey="Chooi, Wai Hon" sort="Chooi, Wai Hon" uniqKey="Chooi W" first="Wai Hon" last="Chooi">Wai Hon Chooi</name></noRegion><name sortKey="Wang, Dong An" sort="Wang, Dong An" uniqKey="Wang D" first="Dong-An" last="Wang">Dong-An Wang</name><name sortKey="Wang, Dong An" sort="Wang, Dong An" uniqKey="Wang D" first="Dong-An" last="Wang">Dong-An Wang</name><name sortKey="Yeo, Suan Siong" sort="Yeo, Suan Siong" uniqKey="Yeo S" first="Suan Siong" last="Yeo">Suan Siong Yeo</name><name sortKey="Zhang, Feng" sort="Zhang, Feng" uniqKey="Zhang F" first="Feng" last="Zhang">Feng Zhang</name><name sortKey="Zhou, Ruijie" sort="Zhou, Ruijie" uniqKey="Zhou R" first="Ruijie" last="Zhou">Ruijie Zhou</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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