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


CONCLUSIONS
When determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.
Fink
Trine
T
Laboratory for Stem Cell Research, Aalborg University, Denmark. trinef@hst.aau.dk
Lund
Pia
P
Pilgaard
Linda
L
Rasmussen
Jeppe Grøndahl
JG
Duroux
Meg
M
Zachar
Vladimir
V
D000199:Actins / Q000235:genetics; D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D002454:Cell Differentiation; D020219:Chondrogenesis; D005260:Female; D020869:Gene Expression Profiling; D005966:Glucuronidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000737:chemistry / Q000166:cytology*; D008875:Middle Aged; D010012:Osteogenesis; D016133:Polymerase Chain Reaction / Q000592:standards*; D012313:RNA / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012015:Reference Standards
D000199:Actins; D012337:RNA, Ribosomal, 18S; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005966:Glucuronidase