TaqMan实时荧光定量逆转录聚合酶链反应检测新型趋化因子巨噬细胞炎症蛋白-2γmRNA表达水平

Detecting a novel CXC chemokine macrophage inflammatory protein-2γ mRNA by real-time fluorescent quantitative reverse transcription polymerase chain reaction (TaqMan)

目的 建立TaqMan实时荧光定量逆转录聚合酶链反应 (RT PCR)法检测新型趋化因子巨噬细胞炎症蛋白 2γ(MIP 2γ)mRNA表达水平 ,并对方法进行评估。方法 TaqMan实时荧光定量RT PCR检测MIP 2γmRNA的表达。结果 线性检测范围达 6个数量级 ,最低检测下限为 6× 10 2 拷贝 ,最高检测上限为 6× 10 7拷贝 ,批间及批内变异 <12 2 %。正常Balb/c小鼠心脏、肝脏、肾脏组织均可检测到MIP 2γmRNA表达 ,以心脏表达水平最高。结论 采用TaqMan实时荧光定量RT PCR检测MIP 2γmRNA表达水平准确、特异、灵敏、快速、操作简便 ,具有临床推广价值。

Objectives To develop and evaluate a real-time fluorescent quantitative RT-PCR method for the detection of a novel CXC chemokine macrophage inflammatory protein-2γ(MIP-2γ)mRNA expression based on TaqMan technique.Method The expression of MIP-2γ mRNA was detected by TaqMan real time fluorscet quantitative RT-PCR. Results The dynamic range of the assay varied from 102 to 107 copies. The intra- and inter-assay coefficient variation (CV) were less than <122%. MIP-2γ mRNA is expressed in heart, liver and kidney of normal Balb/c mice, with the greatest expression in heart.Conclusion The detection of MIP-2γ mRNA expression by real time fluorescent quantitative RT-PCR based on TaqMan technique is more accurate,specific, sensitive and time-saved, which is suitable for use in clinical laboratory.