New validated reference genes for gene expression studies in the differentiating human airway epithelium

Abstract Gene expression values analyzed using RT‒qPCR are usually reported relative to the expression of one or more reference genes (RGs), assumed to be stable regardless of experimental conditions. However, varying cellular conditions, such as cell differentiation, may affect RG expression, compromising the validity and reproducibility of RT‒qPCR studies; thus, it is necessary to determine and validate RG performance in specific cell or tissue types. This study aimed to select suitable RGs for in vitro cultured airway epithelium (AE). The most stably expressed genes were identified using RNAseq and microarray data from AE cultures representing various stages of cell differentiation. The top 15 genes, covering a range of expression levels, were subsequently validated by RT‒qPCR and evaluated using multiple algorithms implemented in the RefFinder tool, and their performance was compared with commonly used universal RGs. A set of genes (including TTC1 , VCP , RHOA , GSS , MGAT4B and AUP1 ) whose expression stability in AE cultures was better than that of traditional RGs was identified. Used as RGs in the analysis of two targets, they provided strikingly consistent results. Identification and rigorous experimental validation of the selected RGs provides a reliable basis for RT‒qPCR studies of AE, which is important for future advancements in respiratory disease research.