Telomeric Repeat Fragment Analysis

TRF analysis was performed as previously described^{65 (link)}. In brief, genomic DNA was isolated by phenol–chloroform extraction and digested with AluI and MboI overnight. A total of 4 µg of digested gDNA was separated on 0.7% agarose gel at 40 V and transferred to a positively charged Nylon membrane (Amersham, RPN203B). After cross-linking the DNA and prehybridization (5× SSC. 0.1% N-lauroylsarcosine sodium salt solution, 0.04% SDS) for 2 h at 65 °C, the membrane was incubated with digoxigenin-labelled TelG probe diluted in hybridization buffer (1.3 nM final concentration) overnight at 65 °C. Digoxigenin-labelled TelG probe was generated as previously described^{66 (link)}. Then, the membrane was washed three times with wash buffer 1 (2× SSC, 0.1% SDS), one time with wash buffer 2 (2× SSC) for 15 min each and blocked in freshly prepared blocking solution (100 mM maleic acid, 150 mM NaCl, pH 7.5, 1% (w/v) blocking reagent (Roche, 11096176001)) for 30 min. Next, the membrane was incubated for 30 min in anti-digoxigenin-AP antibodies (Roche, 11093274910) diluted in blocking solution, washed twice in wash buffer 3 (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.3% (v/v) Tween-20) for 15 min each and equilibrated in AP buffer (100 mM Tris, 100 mM NaCl, pH 9.5) for 2 min. Digoxigenin-labelled telomeric DNA was detected using CDP-star ready to use (Roche, 12041677001) solution.