Telomere Length Measurement by TRF Analysis

Telomere gels were performed using telomere restriction fragment (TRF) analysis. Genomic DNA was digested using AluI and MboI (NEB). 4–10 μg of DNA was run on a 1% PFGE agarose gel (Bio-Rad) in 0.5× TBE buffer using the CHEF-DRII system (Bio-Rad) at 6 V cm⁻¹; initial switch time 5 s, final switch time 5 s, for 16 h at 14 °C. The gel was then dried for 4 h at 50 °C, denatured in a 0.5 N NaOH 1.5 M NaCl solution, and neutralized. Gel was hybridized with ³²P-labelled (CCCTAA)₆ oligonucleotides in Church buffer overnight at 42 °C. The next day, the membrane was washed four times in 4× SSC buffer, exposed onto a storage phosphor screen (GE Healthcare) and scanned using STORM 860 with ImageQuant (Molecular Dynamics). Telomere length was determined using TeloTool software^{35 (link)}.