Telomere Length Measurement by Southern Blot

Telomere length was determined by Southern blotting (TRF analysis) using a TeloTAGGG Telomere Length Assay Kit (Roche) according to the manufacturer’s protocol. Briefly, 1 μg of each DNA sample was digested with Rsa I and Hinf I overnight at 37 °C, electrophoresed on a 0.8% agarose gel at 50 V for 4 h and then transferred to a nylon membrane by Southern blotting. The blotting membrane was blocked and hybridized overnight to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. The washed blot was incubated with anti-DIG-alkaline phosphatase (1:1000 dilution) for 30 min and developed using the substrate in the TeloTAGGG Telomere Length Assay Kit (Roche). The chemiluminescence signals were visualized with a ChemiDoc XRS system (Bio-Rad), and TRF analysis was performed with TeloTool version 1.3 [47 (link)].