细胞IDO基因表达标准化荧光定量PCR检测方法的建立及应用
Establishment and application of a standardized fluorescent quantitative PCR method for detection of cellular IDO gene expression
潘小芬 1闫晗 1康保强 1潘光锦1
作者信息
- 1. 中国科学院广州生物医药与健康研究院 广东 广州 510530
- 折叠
摘要
为了建立一种可以快速准确灵敏直观检测IDO基因表达的方法,通过设计特异性引物,制备IDO的质粒标准品,构建标准曲线,开发了一种实时荧光定量PCR检测方法,应用于IDO表达检测.结果显示,确定IDO基因片段成功克隆至载体,形成重组质粒pSin-flag-IDO,标准曲线呈现良好的线性关系;分别用构建的荧光定量PCR检测方法和作为现有检测标准的普通PCR检测方法进行扩增鉴定,普通PCR最低检测限为660 copies/μL,而荧光定量PCR的最低检测限则低于66 copies/μL,荧光定量PCR检测方法的灵敏度更高;对标准品的5个浓度进行组间重复性检测,变异系数均小于10％,说明检测方法的重复性较好;本方法的建立可使诱导IDO表达检测更准确,有利于人间充质干细胞团体标准的完善.
Abstract
In order to establish a rapid,accurate,sensitive and intuitive method for detecting IDO gene expression,a real-time fluorescence quantitative PCR detection method was developed by designing specific primers,preparing IDO plasmid stan-dards,and constructing a standard curve.The results showed that the IDO gene fragment was successfully cloned into the vector,forming the recombinant plasmid pSin-flag-IDO,and the standard curve exhibited a good linear relationship.Amplifi-cation identification was performed using both the constructed fluorescence quantitative PCR detection method and the con-ventional PCR detection method(as the current detection standard).The lowest detection limit of conventional PCR was 660 copies/µL,while that of fluorescence quantitative PCR was below 66 copies/µL,indicating a higher sensitivity of the fluores-cence quantitative PCR detection method.The inter-group repeatability test was conducted on five concentrations of the standard,with all coefficients of variation being less than 10％,demonstrating good repeatability of the detection method.The establishment of this method can make the detection of induced IDO expression more accurate,which is beneficial for the improvement of the group standards for human mesenchymal stem cells.
关键词
IDO基因表达/荧光定量PCR/检测方法Key words
Gene expression of IDO(Indoleamine 2,3-dioxygenase)/Fluorescent quantitative PCR/Detection method引用本文复制引用
出版年
2024
NETL
NSTL
万方数据