Taqman探针定量检测ICOS/ICOSL基因表达的方法

Quantitative detection of ICOS/ICOSL gene expression using Taqman Probe technology

背景:近年来可诱导共刺激分子(inducible costimulator,ICOS)/可诱导共刺激分子配体(ICOS Ligand,ICOSL)在抗感染、抗移植反应、抗肿瘤、治疗自身免疫性疾病等多方面领域受到了越来越多的关注。目前对ICOS/ICOSL表达量的研究大多基于分子水平,国内外尚鲜见ICOS/ICOSLmRNA定量检测的报道。目的:构建ICOS/ICOSLcDNA质粒标准品,建立Taqman探针检测ICOSL/ICOSL基因表达量的方法。方法:以胃腺癌组织总RNA为模板、Random9mers为引物反转录合成cDNA,用该cDNA为模板聚合酶链反应扩增人ICOS/ICOSL基因相应的cDNA目的片段,构建PMD-18-ICOS/ICOSL重组质粒,鉴定测序后,用Taqman探针实时荧光定量聚合酶链反应制作标准曲线。结果与结论:组织提取的总RNA完整性良好,构建的ICOS/ICOSL质粒测序结果与目的片段一致,质粒的原始浓度为6.10×1013,4.31×1013copies/mL,倍比稀释后用Taqman探针荧光定量聚合酶链反应检测,在1012～1013copies/mL线性关系良好(R2=1),提示成功建立了Taqman探针定量检测ICOS/ICOSL基因表达的方法。

BACKGROUND： The inducible costimulatory molecule （ICOS） / its ligand （ICOSL） become more and more attractive in the field of anti-infection, anti-transplantation reaction, anti-tumor and treatment of autoimmune disease. Current studies focus on molecular level of ICOS/ICOSL gene expression, no study reports quantitative detection of ICOS/ICOSL mRNA expression. OBJECTIVE： To construct the plasmid standard samples of ICOS/ICOSL cDNA, and to detect the ICOS/ICOSL gene expression by the Taqman Probe technology. METHODS： The cDNA was synthesized by reverse transcription with Random 9 mers as the primer and total RNA from the gastric adenocarcinoma tissues as the template. The target sequences of human ICOS/ICOSL cDNA were amplified by polymerase chain reaction amplification from the resulting cDNA as mentioned above. The PMD-18-ICOS/ICOSL recombinant plasmids were constructed. Standard curve was made by Taqman Probe fluorescence polymerase chain reaction technology after gene sequencing. RESULT AND CONCLUSION： The composition of total RNA extracted from the tissues was complete and the sequences of the ICOS/ICOSL plasmid were consistent with the target fragments. The initial concentration of plasmids was 6.10×1013, 4.31×1013 copies/mL. The different diluted concentration of the plasmids had good linear relationship （R2=1） after amplifying by Taqman PCR and the linear range was 1012-1013 copies/mL, suggesting that the method which can quantitatively detect the concentration of ICOS/ICOSL gene by Taqman Probe technology was successfully constructed.