荧光定量聚合酶链反应检测人急性早幼粒细胞白血病PML-RARα融合基因方法的建立

Establishment of real time quantitative-PCR RNA substance for detection of PML-RARα fusion gene of acute promyelocytic leukemia

目的制备荧光定量聚合酶链反应(PCR)方法进行人急性早幼粒细胞白血病(APL)融合基因PMLRARα(bcr1型)mRNA表达量分析的RNA标准品,为白血病微小残留状态(MRD)的监测奠定基础。方法体外转录RNA的方法制备目的基因PMLRARα及管家基因ABL(Ablesongene)RNA标准品。结果目的基因PMLRARα的RNA标准品浓度分别为1×106、1×105、1×104、1×103、1×102拷贝/μl时CT(thresholdcycle,循环阈值,即产生可被检测到荧光信号所需的最少循环数)值分别为20.09、23.58、26.35、29.63、33.48。标准曲线的斜率为-3.30,相关系数为0.999。管家基因ABL标准品浓度分别为1×106、1×105、1×104、1×103、1×102拷贝/μl时,CT值分别为17.27、20.49、24.00、27.28、30.41。标准曲线的斜率为-3.24,相关系数为1.000。结论构建的两个RNA标准品可以得到良好的标准曲线,且标准曲线显示线形关系良好,标准品构建成功。

Objective To establish a real time quantitative-PCR RNA standard substance for detection of PML-RARα fusion gene in acute promyelocytic leukemia. Method The two substances of PML-RARα fusion gene and ABL control gene are from cloning. Results The five successive dilutions（ 1×10^6 ,1×10^5 ,1×10^4 ,1×10^3 ,1× 10^2 copies/μl） of the cloning products of PML-RARα fusion gene were preparedand the CT value were 20. 09,23.58,26. 35,29. 63,33.48. The corresponding standard curve generated aslope of -3.30 and correlation coefficient of 0. 999. The five successive dilutions（ 1×10^6, 1×10^5, 1×10^4 ,1×10^3 ,1×10^2copies/μl） of the cloning products of ABL control gene were prepared and the CT value were17.27,20.49,24. 00,27.28,30. 41. The corresponding standard curve generated a slope of -3.24andcorrelation coefficient of 1. 000. Conclusion We could get good standard curves from the two RNAsubstances. It can be a standard method of real time quantitative-PCR for detection of PML-RARα fusiongene quantitatively in acute promyelocytic leukemia.