Methods for DNA extraction and methylation analysis have been previously described [16 (link)]. Biological age was calculated considering the methylation pattern of 5 CpG sites in five genes (ELOVL2, C1orf132/MIR29B2C, FHL2, KLF14, TRIM59), according to the methodology proposed by Zbieć-Piekarska and colleagues [13 (link)]. The DNA samples (500 ng) were treated with sodium bisulfite using the EZ-96 DNA Methylation-Gold™ Kit (Zymo Research; Irvine, CA, USA). Bisulfite-treated template DNA was then amplified using the GoTaq Hot Start Green Master mix (Promega, Madison, WI, USA). Pyrosequencing was performed with the PyroMark MD System (QIAGEN GmbH, Hilden, Germany). Every sample was tested twice for each gene to guarantee the reproducibility of the experimental setting.
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