实时逆转录聚合酶链反应检测细胞因子表达相对定量方法的建立

Establishment of relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression

目的 建立一种实时逆转录聚合酶链反应 (RT PCR)检测细胞因子表达相对定量方法。方法 以肿瘤坏死因子α(TNFα)为待测基因 ,以葡萄糖 3 磷酸甘油醛脱氢酶 (GAPDH)为内参照进行检测。当靶基因和内参照基因片段克隆入载体后 ,进行纯化和定量及系列稀释 ,建立标准曲线 ,确定实时RT PCR的扩增效率 ;同时优化反应条件 ,使扩增效率接近 10 0 %。然后 ,检测 14份待检标本的TNFα和GAPDH的循环阈值 (Ct)。结果 该法检测的最低拷贝数为 10 3,线性范围为 10 3 ～ 10 9拷贝 ,批内变异系数 (CV)和批间CV分别为 1%和 8%。采用 2 -ΔΔCt法进行数据分析后 ,处理组TNFα的相对表达量比对照组高 8 6～ 9 8倍 ,平均为 9 2倍。结论 新建立的方法敏感 ,重复性好 ,数据处理简便、可靠 。

Objective To establish relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression.Methods TNFα and GAPDH were used as target gene and internal reference respectively. Two gene fragment were cloned, vectors were purified and quantificated. Standard curves were established using a series dilution of quantificated plasmids to measure the amplification efficiency of TNFα and GAPDH. By changing reaction conditions, the amplification efficiency of two gene were nearly 100%. Ct value of TNFα and GAPDH were measured in 14 samples stimuteneously.Results The method can detected as low as 10 3 copies with the linear range was 10 3～10 9 copies, the intra assay and interassay variation was 1% and 8% respectively. TNFα increased 8 6～9 8 fold with the average 9 2 relative to untreated control group analyzed by the 2 -ΔΔCt methods. Conclusions The method we established has fine sensitivity and reproducibility and the data analysis was simple and reliable and can be apply to any genes.