Optimization of gene expression analysis in engineered tissue : determination of proper reference gene for quantitative real-time PCR in 3-d scaffold systems.

• Main Author: Chooi, Wai Hon.
• Other Authors: Wang Dongan
• Format: Final Year Project
• Language: English
• Published: 2010
• Subjects: DRNTU::Engineering::Bioengineering
• Online Access: http://hdl.handle.net/10356/39966
• Institution: Nanyang Technological University

Gene expression study is widely used to obtain information of the cells activities and phenotypes. To quantify gene expression, measurement of the mRNA copy number is commonly done by qualitative RT-PCR. However, reference gene is needed to normalize the expression level of genes of interest (GOI). To interpret the expression level adequately, suitable reference gene need to be determined for different tissues or culture systems. Good reference genes, usually housekeeping genes, are regulated minimally and always expressed stably. In this study, pig synovium-derived mesenchymal stem cells (SMSC) and rabbit chondrocytes were culture in both alginate and agarose scaffolds to set up four different 3D culture systems to form the artificial tissue construct. 7 potential reference genes were selected and compared in these different culture systems, with different set of potential reference genes used in different cell species. The gene expression levels of the tissues were determined by qRT-PCR and then analysed by geNorm and BestKeeper. For pig SMSCs, PPIA and TBP were selected for tissue in alginate scaffold whereas HPRT and TBP were selected for agarose scaffold system. On the other hand, HPRT and RPII were the stable reference genes for rabbit chondrocytes in alginate scaffold while RPII and RPL were selected for rabbit chondrocytes tissue in agarose scaffold.