| 摘要: |
| ［目的］建立荧光实时定量RT-PCR ( FQ RT-PCR)检测Bmi-1基因mRNA的方法并研究该基因在急性白血病细胞中表达的意义。［方法］ 根据 Genbank提供的序列，设计目的基因Bmi-1及内参基因GAPDH的扩增引物，将 Bmi-1及GAPDH RT-PCR扩增片段克隆入载体pMD18-T simple，经测序鉴定正确后，进行纯化、定量及系列稀释，应用 SYBR GreenⅠ荧光染料，建立检测Bmi-1 mRNA的FQ RT-PCR方法，并对其线性及特异性进行评价。应用此方法检测21例急性白血病及10例健康人外周抗凝血中Bmi-1 mRNA的表达水平。［结果］ 该方法的线性范围为1.0×103～1.0×108 copies/μL，相关系数为-1.00，熔解曲线分析扩增产物显示单一的峰，熔解温度(Tm)为80.4℃。急性白血病患者的相对表达量为0.56±0.12，健康对照组中Bmi-1 mRNA的相对表达量为0.19±0.08，两者的表达差异有统计学意义（P<0.05）。［结论］ 实时荧光定量RT-PCR方法检测Bmi-1基因表达，具有高敏感性和高特异性等优点，可作为进一步研究 Bmi-1基因的方法。Bmi-1基因参与急性白血病的发生，可能是一种新的肿瘤标志物。 |
| 关键词: Bmi-1基因 急性白血病 SYBR GreenⅠ 实时荧光定量RT-PCR |
| DOI：10.11724/jdmu.2010.01.24 |
| 分类号:R446；R557 |
| 基金项目: |
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| Expression and significance of Bmi-1 mRNA in acute myeloid leukemia with real-time fluorescent quantitative polymerase chain reaction |
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YU Qi1,2, MENG Xiu-xiang1, YUAN Hong3, WANG Nan3, JIANG Yan-mei3, MA Mo-jiao4
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1.School of Laboratory Medicine, Dalian Medical University, Dalian 116044, China;2.Department of Laboratory Medicine, Taishan Medical College, Tai'an 271016, China;3.The First Affiliated Hospital, Dalian Medical University, Dalian 116011, China;4.No.210 Hospital of PLA, Dalian 116013, China
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| Abstract: |
| ［Objective］ To establish a real-time fluorescent quantitative polymerase chain reaction method for quantifying Bmi-1 mRNA and study its expression and significance in acute myeloid leukemia.［Methods］ The specific primers of Bmi-1 and GAPDH were designed according to GenBank database. The Bmi-1 and GAPDH fragments in pure form from classical RT-PCR were cloned to pMD18-T simple vector respectively, and recombinant plasmids were purified and quantified spectrophotometrically. Using SYBR Green I, a real-time fluorescent quantitative PCR method was established, and its linearity and specificity were evaluated. This method was employed to detect the expression of Bmi-1 mRNA in acute myeloid leukemia and normal controls.［Results］ The linear range was 1.0×103～1.0×108 copies/μL. The relation coefficient was -1.00. Melting curve analysis showed a single peak, and Tm was 80.4℃. The relative expression level of Bmi-1 mRNA in acute myeloid leukemia and normal controls was 0.19±0.08 and 0.56±0.12, respectively (P<0.05).［Conclusions］ SYBR Green I real-time fluorescent quantitative polymerase chain reaction which is specific, sensitive and accurate can be practiced to further research on Bmi-1 gene. Bmi-1 may contribute to leukemogenesis and may be a new tumor marker. |
| Key words: Bmi-1 acute myeloid leukemia SYBR Green I real-time fluorescent quantitative polymerase chain reaction |
