TRF assay was performed to determine telomere length by using the TeloTAGGG Telomere Length Assay Kit (Roche). Briefly, genomic DNA was purified using SDS and proteinase K digestion, followed by phenol-chloroform extraction methods. DNA samples were digested with Hinf I/Rsa I (New England Biolabs). The digested genomic DNA was run on 0.8% gels in 1× tris-acetate-EDTA buffer (6 hours, 5 V/cm). For mouse hepatocyte telomere length analysis, DNA was separated by pulsed-field gel electrophoresis (PFGE, 0.5 to 1.5 s, 6 V/cm) for 20 hours with the λ DNA-Mono Cut Mix (New England Biolabs) as a marker. After electrophoresis, the gels were treated with 0.2 M HCl, denature buffer (0.5 M NaOH and 1.5 M NaCl), and neutralization buffer (0.5 M tris-HCl at pH 8.0 and 3 M NaCl). The DNA were then transferred to nylon membrane by capillary transfer using 20× SSC transfer buffer, hybridized with digoxigenin-telomere probe, and subjected to chemiluminescence detection. The mean TRF length of breast cancer tissues was then calculated as ∑(ODi)/∑(ODi/Li), where ODi is total chemiluminescence above background in interval i and Li is the average length of i in base pairs according to the manufacturer’s instructions.

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