Abstract：
BACKGROUND: The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh.
OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization.
METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking.
RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes.
CONCLUSIONS: RefFinder\'s stability ranking does not consider the influence of primer efficiency.
CONCLUSIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.
OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization.
METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking.
RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes.
CONCLUSIONS: RefFinder\'s stability ranking does not consider the influence of primer efficiency.
CONCLUSIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.
摘要：
背景：RT-qPCR结果的稳健性和可信度关键取决于合适的参考基因的选择。然而,细胞外基质的矿化可以改变细胞内的张力和能量代谢，可能影响传统参考基因的表达，即Actb和Gapdh。
目的：系统地确定合适的参考基因，以研究小鼠成牙骨质矿化。
方法：使用小鼠成牙骨质矿化的时间序列转录组数据。为了确保表达稳定性和中等至高表达水平，在选择潜在的参考基因时采用了3个具体标准.基于DI指数(1/变异系数)对这些基因的表达稳定性进行排序以鉴定前六个潜在的参考基因。对这六个前候选物进行了RT-qPCR验证，将它们的性能与六个以前使用的参考基因(Rpl22，Ppib，Gusb,Rplp0,Actb,和Gapdh）。通过RefFinder分析这12个基因的Cq值以获得稳定性排序。
结果：总共4418个（12.27%）基因符合选择标准。其中,Rab5if,Chmp4b,Birc5,Pea15a,Nudc,Supt4a被鉴定为候选参考基因。RefFinder分析显示，与以前使用的参考基因相比，两个候选基因（Birc5和Nudc）表现出优异的性能。
结论：RefFinder的稳定性排序没有考虑引物效率的影响。
结论：我们建议Birc5和Nudc作为研究小鼠成牙骨质矿化和牙骨质修复的RT-qPCR研究的候选参考基因。
目的：系统地确定合适的参考基因，以研究小鼠成牙骨质矿化。
方法：使用小鼠成牙骨质矿化的时间序列转录组数据。为了确保表达稳定性和中等至高表达水平，在选择潜在的参考基因时采用了3个具体标准.基于DI指数(1/变异系数)对这些基因的表达稳定性进行排序以鉴定前六个潜在的参考基因。对这六个前候选物进行了RT-qPCR验证，将它们的性能与六个以前使用的参考基因(Rpl22，Ppib，Gusb,Rplp0,Actb,和Gapdh）。通过RefFinder分析这12个基因的Cq值以获得稳定性排序。
结果：总共4418个（12.27%）基因符合选择标准。其中,Rab5if,Chmp4b,Birc5,Pea15a,Nudc,Supt4a被鉴定为候选参考基因。RefFinder分析显示，与以前使用的参考基因相比，两个候选基因（Birc5和Nudc）表现出优异的性能。
结论：RefFinder的稳定性排序没有考虑引物效率的影响。
结论：我们建议Birc5和Nudc作为研究小鼠成牙骨质矿化和牙骨质修复的RT-qPCR研究的候选参考基因。
