【摘要】 目的 建立一种用于肝癌早期诊断和监测的血清miRNA-125a Taqman实时荧光定量PCR检测方法。方法 采用Trizol法提取肝癌患者血清中总RNA,以肝癌发生具有代表性的miRNA-125a基因为检测序列,分别设计逆转录引物、扩增引物和Taqman探针;构建和制备miRNA-125a重组质粒标准品,建立血清miRNA-125a Taqman实时荧光定量PCR检测方法。结果 成功构建了miRNA-125a重组质粒载体,制备了miRNA-125a重组质粒标准品,建立了血清miRNA-125a Taqman实时荧光定量PCR扩增体系。经实验验证,所设计引物具有较好的特异性,引物的最低检测限为78.125copies/μl;对17例肝癌患者血清miRNA-125a的检测结果在(1.45×10~2~3.52×10~5)copies/μl之间。结论 血清miRNA-125a Taqman实时荧光定量PCR检测方法的建立,为原发性肝癌早期诊断和监测提供一种新的分子诊断定量方法。更多还原

【Abstract】 Objective Tobuild the method of real-time fluorescence quota PCR examination of serum miRNA-125 a Taqman for diagnosing and monitoring liver cancer early.Methods 1st,based on specific miRNA-125 a geneassociated with liver cancer,to design reverse transcriptase primer,amplification primer and Taqman probeindividually through the Trizaol method to extract patients’ total RNA from serum.2nd,to prepare the miRNA-125 a recombinant plasmid standard substance and buildthe method of real-time fluorescence quota PCR examination of serum miRNA-125 a Taqman.Results To form the miRNA-125 a recombinant plasmid carriers successfully.To prepare the miRNA-125 a recombinant plasmid standard substance successfully.To buildamplification system of real-time fluorescence quota PCR of serum miRNA-125 a Taqman.It had been proved that the designed primer whose minimum detection limit was 78.125 copies/μl had proper specificity.The examination results of serum miRNA-125 a of 17 patients with liver cancer vary between 1.45 × 10~2 ~3.52 × 10~5 copies//μl.Conclusion To set up the method of real-time fluorescence quota PCR examination of serum miRNA-125 a Taqman can offer an efficientmolecular diagnosis way to diagnose and monitor the primary liver cancer early.更多还原