Identification of stable senescence-associated reference genes

Abstract

Cellular senescence is a state of permanent cell cycle arrest activated in response to damaging stimuli. Many hallmarks associated with senescent cells are measured by quantitative real-time PCR (qPCR). As the selection of stable reference genes for interpretation of qPCR data is often overlooked, we performed a systematic review to understand normalization strategies entailed in experiments involving senescent cells. We found that, in violation of the Minimum Information for publication of qPCR Experiments (MIQE) guidelines, most reports used only one reference gene to normalize qPCR data, and that stability of the reference genes was either not tested or not reported. To identify new and more stable reference genes in senescent fibroblasts, we analyzed the Shapiro–Wilk normality test and the coefficient of variation per gene using in public RNAseq datasets. We then compared the new reference gene candidates with commonly used ones by using both RNAseq and qPCR data. Finally, we defined the best reference genes to be used universally or in a strain-dependent manner. This study intends to raise awareness of the instability of classical reference genes in senescent cells and to serve as a first attempt to define guidelines for the selection of more reliable normalization methods.