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

To analyze and compare the performances of two telomere measurement methods (digoxigenin-labeled Southern blot and Real-time PCR) in cellular senescence research.

Genomic 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 optimized. 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.

Digoxigenin-labeled Southern blot system could detect less than 1 μg of human genomic 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 former could distinguish the difference of 2 PDs for 2BS cells while the latter could not distinguish the difference 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).

Digoxigenin-labeled Southern blot system is fully applicable to telomere measurement. 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.