Hinfi and rsai restriction enzymes

The telomere restriction fragment (TRF) assay was performed as previously described (49 (link)) with minor modifications. Briefly, 2.5–7.5 μg of human genomic DNA was digested with HinfI and RsaI restriction enzymes (New England Biolabs) and electrophoresed on a pulse field electrophoresis gel apparatus. HindIII-digested radiolabeled λ-DNA was included in each gel for TL calculation. The gel was hybridized with a (C₃TA₂)₃ probe 5′-labeled with P32 by T4 kinase, overnight at 37°C. Radioactive signal was captured by phosphor screen, which was scanned on a Storm PhosphorImager and quantified using ImageQuant TL software. Mean TRF length was calculated by accounting for the higher signal intensity from larger TRFs, because of multiple hybridization of the telomere-specific hybridization probe, using the formula TRF = ∑(OD_i)/∑(OD_i/L_i), where OD_i is the radioactive signal and L_i is the TRF length at position i.

Telomere restriction fragments were analyzed using the TeloTAGGG Telomere Length Assay kit (Roche). Briefly, cells were harvested by trypsinization, washed in PBS and collected by centrifugation at 400 g for 4 min. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen), digested with HinfI and RsaI restriction enzymes (New England Biolabs) and separated by gel electrophoresis either on 0.8% agarose gels at 50V overnight in 1X TBE buffer or (to resolve elongated telomeres at later time points) on 1% megabase agarose gels (Bio-Rad) using a CHEF DRII equipment (Bio-Rad) under the following conditions: 120° field angle, 5 to 30 s switch times, 5 V/cm and 14°C for 14 hr in 1X TAE. Following the resolution of DNA fragments, DNA was transferred to a positively charged nylon membrane (Roche) by Southern blotting and hybridized with a digoxigenin-labelled telomeric probe. Membranes were exposed to X-ray film (Carestream) and developed in X-OMAT 2000 Processor (Kodak). Mean telomere lengths were calculated as described in Kimura et al. (2010) (link).