颅缝早闭症相关实验荧光定量PCR内参基因的选择研究

Reference Gene Selection for RT-qPCR Gene Expression in Researches of Craniosynostosis

目的探讨用于颅缝早闭症相关实验的荧光定量PCR的最佳内参基因。方法应用与颅骨成骨、颅缝闭合相关的3组实验标本,分别为:Fgfr2cC342Y/+Crouzon综合征小鼠颅缝组织、颅缝早闭患者体外培养颅缝原代细胞和体外培养的小鼠Kusa 4b 10成骨细胞株。以常用的候选管家基因作为研究对象,利用geNorm软件对RT-qPCR结果进行比较分析,以筛选出在不同组织细胞中表达最为稳定的管家基因作为内参。结果Fgfr2cC342Y/+小鼠颅缝组织,Cyc1-Gapdh-Canx为最佳组合;颅缝早闭症患者体外培养颅缝原代细胞,18S rRNA和ATP5B为最佳组合;体外培养的小鼠Kusa 4b10成骨细胞株,18S rRNA和Canx为最佳组合。选择不同内参基因对目的基因RT-qPCR的结果影响显著。结论在设计RT-qPCR实验之前,应针对所选择的标本类型与种类进行管家基因稳定性分析,以增加结果的可信度。

Objective To determine the most stable reference genes for RT-qPCR Gene Expression in Researches of Craniosynostosis. Methods Panels of mouse and human genes were assessed for their stability in the suture tissues of Fgfr2cc3422Y/＋ mouse with Crouzon craniosynostosis syndrome, primary suture cells derived from craniosynostosis patients and Kusa 4b 10 osteoblasts by using the geNorm program. Selected housekeeping genes, individually or in combination, were used to normalise osteocalcin and alkaline phosphatase gene expression data. These genes were considered as reference genes. Results For suture tissues of Fgfr2cc342Y/＋ mouse, Cycl, Gapdh and Canx were in best combination; for primary suture cells derived from craniosynostosis patients, 18S rRNA and ATP5B were in best combination; for Kusa 4b 10 osteoblasts, 18S rRNA and Canx were in best combination. Expression levels and data significance of alkaline phosphatase and osteocalcin were dependent on the reference genes. Conclusion To minimise errors in evaluating gene expression, analysis of the stability of reference genes according to the different sample before RT-qPCR examination is strongly recommended.