内参照基因在实时荧光定量RT-PCR检测中的应用
陈瑾歆;陈建业;李云祥
【期刊名称】《中国老年学杂志》 【年(卷),期】2011(031)014
【摘要】目的 建立测定内参照β-actin表达量的实时荧光定量RT-PCR两步法检测方法.方法 根据Genbank 中人β-actin保守区域序列设计荧光PCR适用的引物和探针,构建质粒标准品建立标准曲线用于荧光PCR相对定量,检测荧光PCR方法的特异性和重复性.结果 建立了人β-actin实时荧光RT-PCR检测方法.结论 本实验建立的人β-actin表达实时荧光RT-PCR两步法检测方法特异性和重复性较好,为β-actin作为定量RT-PCR中内参照基因进行人其他功能基因和病原基因表达的定量分析奠定了基础.%Objective To establish a Taqman real-time RT-PCR 2-step assay for quantitative detection of the expression of human β-actin. Methods Specific primers and probes were designed for real-time RT-PCR according to β-actin cDNA sequence. Plasmid standard preparations were constructed by T-A clone, extracted and prepared for establishing standard curve used for relative quantification real-time RT-PCR. The expression levels of β-actin were measured by real-time RT-PCR in HepG2 cells to detect the specificity and reproducibility of real-time RT-PCR assay. Results An effective real-time RT-PCR assay was established for detecting β-actin mRNA expression levels.Conclusions The real-time PCR assay for the expression of β-actin is a sensitive, specific tool for quantitative assay of
内参照基因在实时荧光定量RT-PCR检测中的应用
