Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

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.