Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. Quantitative method of gene expression analyses via qRT-PCR method provides the accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue obtained from primary and secondary hyperparathyroidism patients and glandular cells isolated enzymatically by the removal of extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We have confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found out that RPLP0>GAPDH>PGK1. Lastly, comparisons of relative target gene (GCM2) expression revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue.

中文翻译：

---

人甲状旁腺组织腺细胞 RT-qPCR 研究的合适内参基因的鉴定

识别合适的参考基因使我们能够了解许多生物过程的根本变化。基因表达分析过程中的标准化对于每种组织/细胞类型都至关重要，包括甲状旁腺组织腺细胞。通过 qRT-PCR 方法进行基因表达定量分析，可准确检测每个目标基因。目前关于人类甲状旁腺组织而非腺细胞类型常用参考基因​​的报道有限。本研究旨在确定并比较甲状旁腺组织细胞最稳定与最不稳定的基因。 43 份取自原发性和继发性甲状旁腺功能亢进症患者的人甲状旁腺组织，以及通过酶法去除细胞外基质成分分离出的腺细胞。提取总RNA后，进行cDNA合成，然后qRT-PCR评估14个候选内参基因。通过 RefFinder 软件（Delta ct、BestKeeper、Genorm 和 NormFinder 算法）确定稳定性，并对五组结果进行评估。即使对不同组进行评估，最稳定的基因是 RPLP0 和 GAPDH，而 CLTC 和 RNA 18S 是最不稳定的。我们通过NormFinder算法单独确认了最稳定的三个基因的综合排名，以了解组间变异，并发现RPLP0>GAPDH>PGK1。最后，相对目标基因 (GCM2) 表达的比较揭示了最稳定的参考基因的相似表达模式。在使用 RefFinder 的四种不同方法的结果评估稳定性的阶段，建议使用最稳定的参考基因。我们希望这项研究能够协助未来的研究，在进行甲状旁腺组织的基因表达分析之前对适当的参考基因进行彻底的评估。