人IL-21 mRNA实时荧光定量PCR检测方法的建立及应用

Establishment and application of the real-time fluorescence quantitative PCR assay for detection of human IL-21 mRNA expression

目的:建立检测人白细胞介素-21(interleukin-21,IL-21)mRNA的实时荧光定量PCR(real-time fluorescencequantitative PCR,qRT-PCR)方法。方法:分离人外周血单个核细胞(peripheral blood mononuclear cells,PBMC),磁珠分选CD4+细胞,经PHA刺激后提取总RNA并逆转录成cDNA。采用qRT-PCR方法检测IL-21 mRNA的表达水平。评价该方法的特异性,应用该方法检测Graves病患者CD4+T细胞中IL-21 mRNA的相对表达水平。结果:建立了人IL-21 mRNA的实时荧光定量PCR检测方法。该法的熔解曲线为单峰,核酸电泳显示为一特异性条带。Graves病患者CD4+T细胞中IL-21 mRNA的相对表达水平明显高于健康对照组(P<0.001)。结论:建立的检测人IL-21 mRNA的实时荧光定量PCR方法灵敏性、特异性好,为进一步临床应用提供了实验基础。

Objective： To establish real-time fluorescence quantitative PCR（qRT-PCR）method for detection of human interleukin-21（IL-21） mRNA expression.Method： The peripheral blood mononuclear cells（PBMC） were separated and CD4＋ cells were isolated using microbeads and stimulated by PHA.Total RNAs were extracted from CD4＋ T cells and then transcribed reversely into cDNAs.The relative expression levels of human IL-21 mRNA in CD4＋ T cells from patients with Graves′disease（GD） and healthy controls were detected by this method.The sensitivity and specificity of the method were evaluated.Results： The melting curve showed a single peak and amplified products were electrophoresed and a single product was detected.The IL-21 mRNA relative expression was significantly increased in GD patients compared to healthy controls（P＆lt;0.001）.Conclusion： The method to detect IL-21 by qRT-PCR was good in specificity.It can be used as a standard method of qRT-PCR for detection of human IL-21 expression.