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

Abstract

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.

Figures

Figure 1

Histochemical staining of adipose-derived stem cells subjected to chondrogenic, osteogenic, or adipogenic induction. The chondrogenic incduction was confirmed by Alcian blue staining of the chondrocyte-specific glucosaminoglycans. The osteogenic phenotype was revealed by staining with Alizarin red, which stains calcium-rich mineral deposits. For the adipogenic cells, intracellular accumulations of lipid droplets were stained with Oil red O.

Figure 2

Determination of least variable reference genes and optimal number of genes for normalization. (A): The average expression stability measure (M) of reference genes during stepwise exclusion of least stable reference gene is shown. The genes are ranked from left to right according to increasing stability. (B): The pairwise variation V between two sequential normalization factors containing an increasing number of genes.

Figure 3

The expression levels of putative reference genes normalized to the geometric mean of YMHAZ, TBP, and GUSB. The values for each gene are relative to the expression level in cells at passage 0 (white bars). The yellow bars indicate subsequent passages, where the increasing intensity corresponds to the duration of culture. The blue bars denote values from chondrogenic cultures and the red indicate those from osteogenic cultures. The two light and dark grey bars represent values from cells cultured for one and two weeks in hypoxia, respectively. Abbreviations: ActB, beta-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; YMHAZ, tyrosine 3/tryptophan 5-monooxygenase activation protein; TBP, TATAA-box binding protein; GUSB, beta-glucuronidase; PPIA, cyclophilin A.

Figure 4

The effect of different normalization factors on the relative gene expression levels of Sox9 and insulin-like growth factor-1 (IGF-1). The expression level of Sox9 is presented after chondrogenic induction relative to values at passage 0. The values have been normalized to either the geometric mean (Geom mean) of tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), and beta-glucuronidase (GUSB), to 18S RNA, or to beta-actin (ActB). The effect of hypoxic culture on expression of IGF-1 is presented after the values have been normalized to either the geometric mean, GAPDH, or ActB levels. Error bars denote standard error of the mean, and asterisks indicate p < .05 compared with values normalized to the geometric mean.