Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells.

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

• Level A · Stronger Clinical Evidence
• Level B · Emerging clinical evidence with positive signals
• Level C · Early human research exploring benefits
• Level D · Scientific groundwork from lab and animal studies
• Emerging · Emerging topic under active research

Abstract (original English)

Background Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative[Formula: see text], RefFinder, and the Livak method. Results Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx an

Evidence level

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

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