Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure

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• 作者：Trine Fink (1)
  Pia Lund (1)
  Linda Pilgaard (1)
  Jeppe Gr?ndahl Rasmussen (1) (2)
  Meg Duroux (1)
  Vladimir Zachar (1)
  
• 刊名：BMC Molecular Biology
• 出版年：2008
• 出版时间：December 2008
• 年：2008
• 卷：9
• 期：1
• 全文大小：438KB
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• 作者单位：Trine Fink (1)
  Pia Lund (1)
  Linda Pilgaard (1)
  Jeppe Gr?ndahl Rasmussen (1) (2)
  Meg Duroux (1)
  Vladimir Zachar (1)
  
  1. Laboratory for Stem Cell Research, Aalborg University, Lyngby, Denmark
  2. Department of Pharmacology, University of Aarhus, 8000, Aarhus C, Denmark
  

文摘

Background For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained. Results In this study, we have compared the gene expression levels of a panel of twelve widely used reference genes during hypoxic culture, osteogenic and chondrogenic differentiation, and passaging of primary human ASCs. We found that several of the commonly used reference genes including 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were unsuitable for normalization in the conditions we tested, whereas tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB) were the most stable across all conditions. Conclusion When determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.