实时逆转录聚合酶链反应定量检测人TNF-α mRNA的表达

Detecting Human TNF-α mRNA by Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

目的建立一种快速、灵敏、特异定量检测人外周血单个核细胞(PBMC)表达 TNF-α mRNA 的方法。方法根据 Gene Bank 中 TNF-α mRNA 序列和 MGB 探针荧光定量分析原理,设计合成特异的引物和探针,优化实验条件,建立定量检测人 TNF-α mRNA 的实时逆转录聚合酶链反应(RT-PCR)方法,并对10名健康体检者 PBMC PHA 刺激前后进行 TNF-α表达检测。结果本法灵敏度为10~3拷贝/毫升、线性检测范围达6个数量级(10~3～10~9拷贝/毫升);特异性好,PCR 产物电泳后无非特异性条带产生,非目的扩增产物用本法进行检测,均无荧光信号响应;以 Ct 值计算变异系数(CV 值),批内批间 CV值均小于5%;10名健康体检者 PBMC PHA 刺激前后 TNF-α mRNA 的平均含量分别为10^(4.15±0.49)和10^(5.19±0.43)拷贝/毫升。结论Taqman-MGB 实时荧光 RT-PCR 定量检测人 TNF-α mRNA 表达水平,结果快速、准确、特异、灵敏,具有临床推广价值。

Objective To establish a rapid specific assay for the quantitative analysis of TNF-α mRNA in peripheral blood mononuelear cells （PBMC） of homo sapiens. Methods According to TNF-α mRNA sequence and the principle of Taqman- MGB probe quantitative assay, the primers and quantitative probes targeted were designed and applied to quantify human TNF -α mRNA by real - time reverse transcription PCR （ RT - PCR） . The specific expression of TNF -α was detected in PBMC of 10 healthy people. Results The optimal system of this method was acquired： the final concentration of fluorescent probe, forward prime, reverse prime and Mg^2 ＋ are 0.1 μmol/L, 0.3μmol/L, 0.3μmol/L and 6 mmol/L respectively. The dynamic range of this assay for TNF -α mRNA was 10^3 - 10^9 copies/mL. No fluorescent signal was detected by real - time fluorescence RT- PCR with non target amplified products. The intra - and inter- run coefficient variation of the CT value were less than 5%. In the 10 heahhy people, the levels of TNF-α mRNA were 10^4.15±0.49 copies/mL before stimulation and 10^5.19±0.43 copies/mL after PHA stimulation. Conclusion The detection of TNF-α mRNA expression by real - time quantitative RT- PCR based on Taqman - MGB technique is more rapid, accurate, sensitive and specific, whieli is suitable for clinical labemtory.