Abstract
BACKGROUND AND OBJECTIVES: Normalization with valid reference genes is crucial for gene expression analysis with quantitative real-time reverse transcription PCR (qRT-PCR). This is especially relevant when stem cells are investigated with respect to gene expression in the differentiation process. Due to the plasticity of the stem cells, the variation of reference gene expression may cause misinterpretation of the target gene expression. METHODS AND RESULTS: In this study, we investigated the gene expression stability of commonly used 32 reference genes in placenta-derived stem cells, which were cultured with or without exogenous epidermal growth factor. The influence of unstable reference gene expression on the data interpretation was also demonstrated with stem cell marker gene expressions on the placenta-derived stem cells. Statistical validation analysis of reference genes revealed the stability of each gene. Commonly used β-actin, 18S and GAPDH expression were relatively instable. The cell cycle relating house keeping genes, PPIA, POLR2A, and POP4 were most stable in the compared culture conditions. Reference genes were divided into the following three groups and statistically analyzed; 1) unstable genes, 2) stable genes, and 3) commonly used genes. The results indicate that the interpretation of the experiments was significantly different depending on the stability of the reference genes. CONCLUSIONS: In the stem cell experiments, even minor differences in the culture conditions influenced the expression of reference genes. Thus, the identification of valid reference genes must be determined at each experimental setting. We recommend performing a stepwise screening process to determine valid reference genes.