摘要
目的建立用实时定量逆转录聚合酶链反应(RT-PCR)检测急性早幼粒细胞白血病(APL)PML-RARα mRNA的方法。方法制备目的基因PML-RARα的RNA标准品及管家基因-βactin的RNA标准品。然后将目的基因PML-RARα和管家基因-βactin的标准品梯度稀释作为模板进行实时荧光定量PCR反应,制作标准曲线。利用成功构建的两个标准曲线测定APL患者的目的基因PML-RARα与管家基因-βactin的相对值,分析不同状态患者的微小残留病(MRD)水平是否有差别,从而指导临床的治疗。结果用本研究建立的实时定量RT-PCR方法可检测出10～5μg NB4细胞cDNA中的PML-RARα融合基因,其重复性的循环域值(Ct值),管间、批间变异系数(CV)分别为2.2%、4.0%。结论本研究所建立的实时定量RT-PCR方法灵敏度高、重复性好。应用本法检测急性早幼粒细胞白血病PML-RARα融合基因表达水平的改变,有助于监测白血病微小残留病,评价疗效及判断预后。
Objective To set up the method of testing acute promyelocytic leukemia（APL） PML-RARα fusion gene by Real-time Quantitative RT-PCR.Methods We prepared RNA standards of the target gene of PML-RARα and the house-keeping gene of β-actin respectively,and then prepared serial dilutions of PML-RARα gene and housekeeping gene β-actin followed by real-time fluorescent quantitative PCR reaction,based on which standard curves were plotted.Using these two standard curves,we tested the relative levels of PML-RARα and β-actin genes and analyzed whether minimal residual disease（MRD） levels from patients at different states have discrepancy.Results Using the method of real-time quantitative RT-PCR we set up in this study,we could test PML-RARα fusion gene from 10～5 μg cDNA of NB4 cells and the corresponding repetitive Ct values.Coefficients of variation（CV） between tubes and between batches were 2.2% and 4.0% respectively.Conclusion The method of real-time quantitative RT-PCR we set up in this study has high sensitivity and good reproducibility.In addition,this method has great significance in monitoring MRD and evaluating efficacy and prognosis by testing APL PML-RARα fusion gene.
出处
《检验医学与临床》
CAS
2011年第14期1669-1671,共3页
Laboratory Medicine and Clinic
基金
沈阳市科技局科学技术项目资助项目(F10-218-1-00)
