Department of Endodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 401147, P.R. China.
Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147, P.R. China.
Int J Mol Med. 2022 Nov;50(5). doi: 10.3892/ijmm.2022.5188. Epub 2022 Sep 7.
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 (, , , , , , , , , , and ) 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. exhibited the highest stability in the SMG developmental stage, while 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.
唾液对于润滑口腔和辅助食物消化至关重要。然而，口腔功能障碍的发生，如口干症、吞咽困难或口腔感染，会显著降低受影响个体的生活质量。主要的唾液腺包括颌下腺（SMG）、舌下腺和腮腺；它们是哺乳动物中较大的腺体，产生大部分唾液。SMG 是研究分支形态发生和功能再生的有效模型。为了更好地理解唾液腺发育和功能再生过程中的关键动态基因表达模式，寻找一组可靠的参考基因至关重要。因此，本研究旨在确定 SMG 发育和功能再生状态下用于基因表达数据标准化的优异参考基因。首先，从胚胎期和新生期的小鼠中采集发育中的 SMG 样本。在几个阶段从导管结扎/松解模型中获得功能再生样本。通过转录组测序（RNA-seq）分析，从涉及微管蛋白、组蛋白、肌动蛋白、泛素和 GAPDH 家族成员的 430 个候选物中筛选了 12 个参考基因（、、、、、、、、、和）。然后使用 RT-qPCR（SYBR-Green）和 Western blot 分析半定量评估基因和蛋白质表达。使用ΔCq、geNorm、BestKeeper、NormFinder 和 RefFinder 方法和软件评估表达稳定性。在 SMG 发育阶段，表现出最高的稳定性，而在 SMG 再生阶段，被推荐为最稳定的参考基因。总之，本研究为 SMG 在发育和功能再生阶段提供了参考基因的选择依据。
