Telomere restriction fragment (TRF) analysis by Southern blot

TRF analysis was performed as previously described [54]. Briefly, genomic DNA was extracted from freshly isolated tissue using lysis buffer (Fermentas K0512) supplemented with 1 mg/ml Proteinase K (Sigma, MO, USA) and RNase A (1:100 dilution, Sigma, MO, USA). Samples were incubated at 50°C for 18 h in a thermomixer and genomic DNA was extracted by equilibrated phenol-chloroform (Sigma, MO, USA) and chloroform-isoamyl alcohol extraction (Sigma, MO, USA). Genomic DNA was quantified and normalized so the same amount of DNA was digested with RSAI and HINFI enzymes (NEB, MA, USA) for 12 h at 37°C. Samples were electrophoresed on a 20 cm 0.6% agarose gel, in 0.5% TBE buffer, at 4°C for 17 h at 110 constant voltage. A 1.6 kb telomere probe, (TTAGGG)n, labelled with [α-32P]-dCTP using the Prime-it II random primer labelling kit (Stratagene) was used for Southern blotting. 3–6 different WT and tert^-/- individuals belonging to different age groups were used for quantification of each Southern Blot experiment.