Analysis of the stability of 70 housekeeping genes during iPS reprogramming

Abstract

Studies on induced pluripotent stem (iPS) cells highly rely on the investigation of their gene expression which requires normalization by housekeeping genes. Whether the housekeeping genes are stable during the iPS reprogramming, a transition of cell state known to be associated with profound changes, has been overlooked. In this study we analyzed the expression patterns of the most comprehensive list to date of housekeeping genes during iPS reprogramming of a mouse neural stem cell line N31. Our results show that housekeeping genes’ expression fluctuates significantly during the iPS reprogramming. Clustering analysis shows that ribosomal genes’ expression is rising, while the expression of cell-specific genes, such as vimentin (Vim) or elastin (Eln), is decreasing. To ensure the robustness of the obtained data, we performed a correlative analysis of the genes. Overall, all 70 genes analyzed changed the expression more than two-fold during the reprogramming. The scale of this analysis, that takes into account 70 previously known and newly suggested genes, allowed us to choose the most stable of all genes. We highlight the fact of fluctuation of housekeeping genes during iPS reprogramming, and propose that, to ensure robustness of qPCR experiments in iPS cells, housekeeping genes should be used together in combination, and with a prior testing in a specific line used in each study. We suggest that the longest splice variants of Rpl13a, Rplp1 and Rps18 can be used as a starting point for such initial testing as the most stable candidates.

Housekeeping Genes for Parkinson’s Disease in Humans and Mice

“…The data obtained indicate that there is no universal HKG both for different brain tissues of one organism and for similar tissues of different organisms. This conclusion is supported by other studies: researchers must select and validate a set of reference genes for each specific cell line [ 41 ].…”

“…However, in 2020, the same group of scientists, when analyzing HKGs in induced pluripotent stem cells of the same lineage, concluded that GAPDH has a highly variable expression during reprogramming. This gene is not attractive as a reference gene for the analysis of expression in works related to the process of fibroblast reprogramming [ 41 ]. The authors point out that the way the primers were selected changed from the first publication to the second.…”

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue

“…Our analyses showed that different reference genes revealed no stable expression during the development of the human inner ear. Using one single reference gene is generally not recommended, as discussed in Vandesompele et al (2002) [57], and Panina et al (2020) [58]. Normalization against one reference gene may lead to misleading results.…”

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

“…Phosphoglycerate mutase 1 (PGAM-1) expresses an enzyme responsible for catalyzing 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA), playing an essential role in glycolysis. It is also ubiquitously distributed in multiple cell types [ 52 , 53 ]. Shared characteristics related to vital processes in the organism, such as glycolysis, immune response, protein folding and cell structure, especially due to applicability to multiple species and most tissues, bring up those 6 genes as relevant targets to investigate as suitable reference genes in our study.…”