Validation of superior reference genes in mouse submandibular glands under developmental and functional regeneration states

Saliva is crucial for lubricating the mouth and aiding in food digestion. However, the occurrence of oral dysfunction, such as xerostomia, dysphagia or oral infection can markedly reduce the quality of life of affected individuals. The major salivary glands include the submandibular gland (SMG), and sublingual and parotid glands; they are the larger glands in mammals that produce the majority of the saliva. The SMG serves as an effective model for the study of branching morphogenesis and functional regeneration. In order to better understand the key dynamic gene expression patterns during salivary gland development and functional regeneration, it is crucial to search for a panel of reliable reference genes. The present study thus aimed to identify superior reference genes to normalize gene expression data in the SMG under states of development and functional regeneration. First, the developmental SMG samples were harvested from mice in the embryonic and post-natal periods. Functional regeneration samples from a ductal ligation/de-ligation model were obtained at several stages. A total of 12 reference genes (Actb, Actg1, Ubc, Uba1, Uba52, Ube2c, Tuba1a, Tuba1b, Tubb5, H2afy, H2afx and Gapdh) from 430 candidates involving tubulin, histone, actin, ubiquitin and GAPDH family members were screened via transcriptome sequencing (RNA-seq) analysis. RT-qPCR (SYBR-Green) and western blot analysis were then used to semi-quantitatively assess gene and protein expression. The stability of expression was evaluated using the ΔCq, geNorm, BestKeeper, NormFinder and RefFinder methods and software. Actg1 exhibited the highest stability in the SMG developmental stage, while Tubb5 was recommended as the most stable reference gene for the SMG regenerative stage. In summary, the present study provides evidence-based selections for superior reference genes in the SMG during the stages of development and functional regeneration.