Abstract
Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.
Highlights
Aging is a complex physiological process that affects organismal, tissue and cellular levels, and it is characterised by a persistent loss of cellular and tissue integrity that leads to impaired biological function and increased risk of pathologies and diseases[1]
The first model examined was the oncogene-induced senescence (OIS) model, which was established in immortalised primary human BJ foreskin fibroblasts (BJ fibroblasts) transduced with the H-RAS oncogene fused to a 4-hydroxytamoxifen (4-OHT)-responsive Estrogen Receptor (ER) ligand binding domain[39]
The second model employed was the in vitro aging model, which consists of the serial culture of primary human dermal fibroblasts (HDFs), allowing the analysis of gene expression in a constant environment over time[40]
Summary
Aging is a complex physiological process that affects organismal, tissue and cellular levels, and it is characterised by a persistent loss of cellular and tissue integrity that leads to impaired biological function and increased risk of pathologies and diseases[1]. Studies using cells aged in vitro after extensive passaging have been widely used in aging research. Cells derived from old individuals share cellular and molecular phenotypes with in vitro senescent cells, but it is not clear whether these phenotypes are completely overlapping[8] For this reason, when possible, researchers use in vivo model studies in which cells from young and elderly donors are used immediately after obtention or after a few passages in culture. These are the currently used models or approaches to study aging, careful consideration must be given to the differences among them. Identification of appropriate reference genes is a crucial step in the correct development of RT-qPCR assays
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