TGF-β_1mRNA荧光实时定量PCR检测初筛方法的建立及初步运用  

Establishment and preliminary application of the screening model for transforming growth factor β_1 mRNA by fluorescence quantitative reverse transcription polymerase chain reaction

作　　者：胡璟谊[1] 李君[2] 季敬璋[3] 吕建新[3] 

机构地区：[1]温州市第二人民医院检验科,浙江温州325000 [2]温州市第三人民医院检验科,浙江温州325000 [3]温州医学院检验医学院细胞与分子医学研究所,浙江温州325035

出　　处：《温州医学院学报》2006年第4期309-312,共4页Journal of Wenzhou Medical College

基　　金：浙江省自然科学基金资助项目(397510)。

摘　　要：目的:建立荧光实时定量检测人转化生长因子异构体(TGF-β1)mRNA的方法。方法:抽取外周静脉血,予EDTA抗凝,用淋巴细胞分离液分离外周血单个核细胞,用Trizol裂解液提取总RNA,将总RNA逆转录为cDNA,在扩增体系中加入SYBRGreenⅠ,应用FQ-RT-PCR定量检测TGF-β1mRNA。结果:该法检测TGF-β1mRNA的灵敏度达105拷贝/ml,线性范围为105～1011拷贝/ml,Ct值的批内、批间变异系数分别为2.27%～2.56%和4.78%～5.22%。临床应用显示,糖尿病患者PBMC中TGF-β1mRNA的含量显著高于正常对照组(P<0.05)。结论:FQ-RT-PCR应用于检测TGF-β1mRNA的初筛方法具有简便、快速等特点,为临床应用提供了方法学依据。Objective： To develop fluorescence reaction （FQ-RT-PCR） for quantitative detection quantitative reverse transcription polymerase chain of transforming growth factor β1 （TGF-β1 ） mRNA in peripheral blood mononuclear cell of patients with diabetes mellitus. Methods：the PBMC was separated, and the total RNA was extracted with Trizol lysate, and then the cDNA was transcripted, the SYBR GREEN-I was used in the amplification system for quantitative detection of TGF-β1, mRNA. Results：The FQ-RT-PCR could detect as low as 10^5 copies per milliliter recombined plasmid standard,the linear range was from 10^5 to 10^11 copies per milliliter, and the coefficient variation were 2.27%-2.56% and 4.78%- 5.22% in intra and inter-assay respectively. The levels of TGF-β1, mRNA in PBMC of patients with diabetes mellitus were significantly higher than that of healthy controls （P〈0.05）.Conclusion： FQ-RT-PCR is a simple and rapid method as the primary application of the screening model for transforming growth factor β1 mRNA that eliminates the post-PCR processing steps.

关 键 词：逆转录-聚合酶链反应 转化生长因子-Β1 糖尿病 

分 类 号：R512.62[医药卫生—内科学] R575.2[医药卫生—临床医学]