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.

Figure 1

Box-and-whisker plot indicating range of Ct values of candidate reference genes throughout iPS reprogramming. Values of three biological replicates taken as averages of 4 technical replicates are given. The whiskers represent standard deviation of n samples (n = 24).

Figure 2

Expression profile of the 12 candidate reference genes throughout the 20 days of the reprogramming process. Measurements were performed in triplicate for each day. For each gene, linear fits were applied (black lines) and the displayed grey areas represent the 95% confidence intervals. For visualization purpose, we added a color bar representing the log2 values of Cycle threshold.

Figure 3

The impact of the choice of different reference genes on the target gene expression of three pluripotent markers. Fold change values of three pluripotency markers, Nanog, Oct4 and Rex1, were calculated using Atp5f1 (full line, filled circles) Rps18 (dashed line, white circles) and Gapdh (dotted line, filled squares). When using Atp5f1 as a reference gene, which was selected as the best candidate housekeeping gene, the fold change values greatly increased over time, corresponding to an increase in the expression of Nanog, Oct4 and Rex1. When using Rps18 as a reference gene, which was selected as the least stable gene during reprogramming, very low values of gene expression were obtained, without any increase during the later stages of reprogramming. Gapdh, a candidate reference gene ranked 2nd, 3rd or 4th among all tested genes, produced an increase in expression patterns of pluripotency genes, however, to a lower extent than the gene Atp5f1.