TaqMan实时荧光定量RT-PCR检测外周血单个核细胞TRAIL-mRNA  

Detection of TRAIL mRNA of mononuclear cell in peripheral blood by TaqMan real-time fluorescence quantitative RT-PCR

作　　者：毛丽萍[1] 王惠民[2] 张子玉[1] 吴月平[1] 章幼奕[1] 鞠少卿[2] 陈育凤[1] 

机构地区：[1]南通大学附属南通第三医院检验科,江苏南通226006 [2]南通大学附属医院检验科,南通226001

出　　处：《临床检验杂志》2007年第3期207-209,共3页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省南通市社会发展基金资助课题(S2006043)。

摘　　要：目的 建立TaqMan实时荧光定量RT-PCR（rQ-RT-PCR）检测外周血单个核细胞（PBMC）中肿瘤坏死因子相关的凋亡诱导配体（TRAIL）mRNA含量的方法,探讨PBMC中TRAIL mRNA的检测价值.方法 在TRAIL基因第二和第三外显子区设计特异性引物和荧光探针,用RT-PCR扩增目的片段,实时检测产物的荧光强度,以标准品建立标准曲线,软件自动计算出待测样本中TRAIL mRNA含量,以TRAIL mRNA和β2M mRNA含量的对数比值进行TRAIL mRNA表达水平评价.结果 TRAIL mRNA含量线性范围为（2.9×10^3～2.9×10^9）copies/ml,批内和批间变异系数分别为9.5%和16.7%.30例健康献血员和58例慢性乙肝患者TRAIL mRNA与β2M mRNA浓度的对数比值的（x）±s分别为0.528±0.014和0.775±0.038,两组差异显著,P＜0.001.结论 FQ-RT-PCR检测TRAIL mRNA水平具有较好的灵敏度和重复性,适用于临床研究.Objective To establish and evaluate the method using real-time fluorescence quantitative polymerase chain reaction based on TaqMan technique for detecting TNF-related apoptosis inducing ligand （TRAIL） mRNA in mononuclear cell （PBMC） of human peripheral blood. Methods The probe and specific primers （ upper and lower） were designed according to the sequences of the second and the third extron of the TRAIL gene respectively. The fluorescence of PCR products was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression levels of TRAIL mRNA in clinical samples were determined with software and the results presented as the ratios of TRAIL mRNA to β2M mRNA. Results The detection range of the assay was from 2.9 × 10^3 to 2.9 × 10^9 copies/ml. The coefficients of variation （CV） of intra-experimental and inter-experimental reproducibility were 9.5% and 16.7% respectively. The ratios of TRAIL mRNA to β2M mRNA in patients with chronic hepatitis B were significantly higher than those of blood donors （P 〈0.001 ）. Conclusion The assay established in this study presented high sensitivity and reproducibility, so it is suitable for measurement of the expression level of TRAIL-mRNA

关 键 词：肿瘤坏死因子相关的凋亡诱导配体 单个核细胞 实时荧光定量RT-PCR 

分 类 号：Q503[生物学—生物化学]