Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models

Wai Hon Chooi; Ruijie Zhou; Suan Siong Yeo; Feng Zhang; Dong-An Wang

doi:10.1007/s12033-012-9604-x

Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models

Wai Hon Chooi, Ruijie Zhou, Suan Siong Yeo, Feng Zhang, Dong-An Wang^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

14 Citations (Scopus)

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. © 2012 Springer Science+Business Media New York.

Original language	English
Pages (from-to)	623-633
Journal	Molecular Biotechnology
Volume	54
Issue number	2
DOIs	https://doi.org/10.1007/s12033-012-9604-x
Publication status	Published - Jun 2013
Externally published	Yes

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Publication details (e.g. title, author(s), publication statuses and dates) are captured on an “AS IS” and “AS AVAILABLE” basis at the time of record harvesting from the data source. Suggestions for further amendments or supplementary information can be sent to [email protected].

Research Keywords

3D culture
Chondrocytes
Hydrogel
Mesenchymal stem cells
Quantitative PCR
Reference gene
Tissue engineering

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title = "Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models",

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. {\textcopyright} 2012 Springer Science+Business Media New York.",

keywords = "3D culture, Chondrocytes, Hydrogel, Mesenchymal stem cells, Quantitative PCR, Reference gene, Tissue engineering",

author = "Chooi, {Wai Hon} and Ruijie Zhou and Yeo, {Suan Siong} and Feng Zhang and Dong-An Wang",

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Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models. / Chooi, Wai Hon; Zhou, Ruijie; Yeo, Suan Siong et al.
In: Molecular Biotechnology, Vol. 54, No. 2, 06.2013, p. 623-633.

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

TY - JOUR

T1 - Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models

AU - Chooi, Wai Hon

AU - Zhou, Ruijie

AU - Yeo, Suan Siong

AU - Zhang, Feng

AU - Wang, Dong-An

N1 - Publication details (e.g. title, author(s), publication statuses and dates) are captured on an “AS IS” and “AS AVAILABLE” basis at the time of record harvesting from the data source. Suggestions for further amendments or supplementary information can be sent to [email protected].

PY - 2013/6

Y1 - 2013/6

N2 - 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. © 2012 Springer Science+Business Media New York.

AB - 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. © 2012 Springer Science+Business Media New York.

KW - 3D culture

KW - Chondrocytes

KW - Hydrogel

KW - Mesenchymal stem cells

KW - Quantitative PCR

KW - Reference gene

KW - Tissue engineering

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DO - 10.1007/s12033-012-9604-x

M3 - RGC 21 - Publication in refereed journal

C2 - 23054629

SN - 1073-6085

VL - 54

SP - 623

EP - 633

JO - Molecular Biotechnology

JF - Molecular Biotechnology

IS - 2

ER -

Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models

Abstract

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Research Keywords

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