Abstract
Induced pluripotent stem cell (iPS) reprogramming allows to turn a differentiated somatic cell into a pluripotent cell. This process is accompanied by many changes in fundamental cell properties, such as energy production, cell-to-cell interactions, cytoskeletal organization, and others. Real-time quantitative polymerase chain reaction (RT-qPCR) can be used as a quantitative method of gene expression analysis to investigate iPS reprogramming but it requires a validation of reference genes for the accurate assessment of target genes’ expression. Currently, studies evaluating the performance of reference genes during iPS reprogramming are lacking. In this study we analysed the stability of 12 housekeeping genes during 20 days of iPS reprogramming of murine cells based on statistical analyses of RT-qPCR data using five different statistical algorithms. This study reports strong variations in housekeeping gene stability during the reprogramming process. Most stable genes were Atp5f1, Pgk1 and Gapdh, while the least stable genes were Rps18, Hprt, Tbp and Actb. The results were validated by a proof-of-point qPCR experiment with pluripotent markers Nanog, Rex1 and Oct4 normalized to the best and the worst reference gene identified by the analyses. Overall, this study and its implications are particularly relevant to investigations on the cell-state and pluripotency in iPS reprogramming.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful technique that allows to monitor relative changes in gene expression and is considered the “gold standard” in the field of mRNA quantification[10]
Using pluripotency markers Nanog, Rex[1] and Oct[4] whose levels are known to rise during reprogramming, we further demonstrated that choosing Rps[18] as opposed to Atp5f1 would lead to inadequate normalization
It is known that these alterations include most basic, “housekeeping” functions such as cell metabolism[4], speed of the cell cycle[9] and lipid profile[26,27]
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful technique that allows to monitor relative changes in gene expression and is considered the “gold standard” in the field of mRNA quantification[10]. This technique requires a normalization strategy to ensure the reliability of the data[11,12]. Recent works have uncovered that housekeeping genes’ expression levels may vary depending on the gene, cell type and experimental conditions. The RT-qPCR analysis of common housekeeping genes’ stability over the time course of iPS reprogramming has never been performed. Implications of our results are discussed in the context of iPS biology
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