实时荧光定量RT-PCR检测糖皮质激素α-受体mRNA表达水平方法的建立  

Development of the standard curve for detecting the expression of human glucocorticoid receptor-α mRNA with real time fluorescence quantitative RT-PCR

作　　者：熊涛[1] 曹政[1] 

机构地区：[1]长江大学生命科学学院,湖北荆州434025

出　　处：《湖北大学学报（自然科学版）》2008年第3期294-297,共4页Journal of Hubei University：Natural Science

基　　金：湖北省教育厅青年资助项目(Q200712005);长江大学博士启动基金(0364)资助项目

摘　　要：建立用荧光定量RT-PCR的方法检测人糖皮质激素α-受体(GR-α)mRNA水平的标准曲线.根据人糖皮质激素α-受体mRNA序列(GeneBank No.NM_000176)设计引物,反转录出cDNA,再插入到PMD18-T载体中.再根据cDNA设计引物和探针,采用Taq Man探针荧光定量RT-PCR的检测方法,构建人GR-α基因表达量的标准曲线.由PMD18-T-GR-α所构建的标准曲线线性关系良好(反应体系中含1×101-1×105拷贝数的人GR-α基因分子时,扩增反应Ct值与拷贝数的对数呈较好的线性关系)、灵敏度高(可检测出低于10个拷贝/μL的样品)、特异性强、准确可靠.The standard curve for detecting human glucocorticoid receptor alpha （GR-α） mRNA expression with fluorescence quantitative polyrnerase chain reaction was developed. The probe and primers were designed and synthesized according to the human GR-α （GeneBank No. NM 000176） mRNA sequence. Then a TaqMan probe fluorescence quantitative RT- PCR assay was carried out, and the standard curve for detecting human GR-a mRNA expression was constructed. The standard curve made by pMD18 - T - GR-α had good linear dependence, and it was sensitive （it could detect 〈 10 copies/μL of plasmid DNA） and specific. The standard curve for detecting human GR-amRNA expression with fluorescence quantitative RT-PCR was developed successfully.

关 键 词：TAQMAN探针 荧光定量RT—PCR 标准曲线 

分 类 号：Q785[生物学—分子生物学] S565.403.2[农业科学—作物学]