Screening and validation of stable housekeeping genes in lung organoids and rat lung models under tobacco exposure

In recent years, with the increasing severity of air pollution and environmental degradation, research on lung-related diseases has become more intensive. Lung organoids, as 3D <i>in vitro</i> culture models, can simulate the local microenvironment and physiological functions of lung tissue and are widely used in studies on the development and mechanisms of lung-related diseases. However, the precise application of lung organoids is still in the developmental stage, particularly regarding the screening and validation of stable housekeeping genes in lung organoids, which remains unclear. This study utilized human/mouse-derived lung organoids and rat lung tissue as research subjects. By establishing physiological, traditional cigarette, and heated cigarette exposure models and combining BestKeeper, GeNorm, and NormFinder software, the expression stability of various housekeeping genes under different research subjects and exposure models was analyzed to identify stable housekeeping genes for lung-related research. The results showed that in human/mouse-derived lung organoids and rat lung tissue, the baseline expression levels of housekeeping genes were generally high. Among them, GAPDH exhibited the highest expression stability and was least affected by exposure environments, followed by β-actin, RPS16, and RPL19, while 18s showed relatively poor stability. Furthermore, when using a stable single housekeeping gene (e.g., GAPDH) for relative quantification of target gene expression, the experimental results were more significant. When GAPDH and β-actin were used as combined housekeeping genes for target gene quantification, the changes in target gene expression were more pronounced, with stronger statistical significance. In conclusion, this study provides stable single housekeeping genes (GAPDH) and combined housekeeping genes (GAPDH + β-actin) for lung organoid research, contributing to further advancements in the study of lung health.