DNA印迹法和实时定量PCR法在端粒长度测定中的应用比较

Comparison of Southern blotting and Real-time PCR in measuring telomere shortening

目的:分析并比较测量端粒长度的DNA印迹法和实时定量PCR法在细胞衰老实际研究中的性能。方法:从不同代数(population doublings,PDs)的正常人成纤维细胞(2BS)中提取基因组DNA作为测试样本;对测量端粒长度的DNA印迹法和实时定量PCR法进行优化,以点杂交的方式分析地高辛配基标记检测系统的特异性和灵敏度;分别用两种方法反复测定同一样本的平行样或不同样本并分析比较两种方法测量端粒的分辨率和精确度。结果:实际测量端粒时,地高辛配基标记检测系统虽可检测出小于1μg的基因组DNA,但最优样本量为4～5μg;DNA印迹法的分辨率约为150 bp,实时定量PCR法的分辨率约为300～400 bp,前者可分辨代龄相差少至2 PDs的2BS细胞端粒长度的差异,而后者只能分辨代龄相差5 PDs以上的端粒差异;实时定量PCR法重复测量误差超过10%,远大于DNA印迹法的2.5%(P<0.001)。结论:地高辛配基标记的DNA印迹检测系统完全适用于测量端粒长度,且性能优于实时定量PCR法,但后者方便快捷,可高通量处理样本,所以在细胞衰老研究中,应根据具体情况合理选择相应方法。

Objective ：To analyze and compare the performances of two telomere measurement methods （digoxigenin-labeled Southern blot and Real-time PCR） in cellular senescence research. Methods： Ge-nomic DNA extracted from normal human fibroblasts （2BS） of different population doublings （PDs） was used as test samples. The Southern blot and Real-time PCR methods for telomere measurements were op-timized. The specificity and sensitivity of digoxigenin detection system were analyzed by dot blot. The two methods were respectively used to measure parallel samples to analyze and compare their resolution and accuracy. Results： Digoxigenin-labeled Southern blot system could detect less than 1 μg of human ge- nomic DNA, but the optimal sample size was around 4-5 μg when measuring telomeres. The resolution of the Southern blot method was around 150 bp while the Real-time PCR method 300-400 bp. The for-mer could distinguish the difference of 2 PDs for 2BS cells while the latter could not distinguish the differ-ence of less than 5 PDs. The measurement error of the repeated measurements for the Real-time PCR method was more than 10% which was bigger than that of the Southern blot method （2.5% , P 〈 0.001 ）. Conclusion： Digoxigenin-labeled Southern blot system is fully applicable to telomere measure-ment. The performance of the Southern blot method is better than that of the Real-time PCR method while the latter is convenient and high-throughput. In the study of cellular senescence, the appropriate method should be selected according to specific experiment.