Molecular Biotechnology

, Volume 54, Issue 2, pp 623–633 | Cite as

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 WangEmail author


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.


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


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Wai Hon Chooi
    • 1
  • Ruijie Zhou
    • 1
  • Suan Siong Yeo
    • 1
  • Feng Zhang
    • 1
  • Dong-An Wang
    • 1
    Email author
  1. 1.Division of Bioengineering, School of Chemical and Biomedical EngineeringNanyang Technological UniversitySingaporeRepublic of Singapore

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