DNA Extraction and Methylation Analysis

Whole blood samples, collected into EDTA tubes from each participant, were centrifuged at 2500 rpm for 15 min. The buffy coat fraction was transferred to a cryovial and immediately frozen at −20°C until use. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Italy) according to the manufacturer’s protocol. LINE-1 methylation levels were measured by pyrosequencing-based methylation analysis, using the PyroMark Q24 instrument (Qiagen, Italy), as previously reported (Agodi et al., 2015a; Barchitta et al., 2017c). Briefly, bisulfite conversion and clean-up of DNA for methylation analysis of 30–40 ng of DNA were completed using the EpiTect Bisulfite Kit (Qiagen, Italy) and the converted DNA was eluted in 20 μl of Elution Buffer. PCR was conducted in a reaction volume of 25 μl, using the PyroMark PCR Kit (Qiagen, Italy). According to the manufacturer’s instructions, each reaction mixture contained 1.5 μl of bisulfite-converted DNA, 12.5 μl of PyroMark PCR Master Mix 2×, 2.5 μl of Coral Load Concentrate 10×, and 2 μl of the forward primer (5′-TTTTGAGTTAGGTGTGGGATATA-3′) and the reverse-biotinylated primer (5′-biotin-AAAATCAAAAAATTCCCTTTC-3′) (0.2 μM for each) (Agodi et al., 2015b). Hot start PCR cycling conditions were 1 cycle at 95°C for 15 min, 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. Then, the PCR product underwent pyrosequencing using 0.3 mM of the sequencing primer (5′-AGTTAGGTGTGGGATATAGT-3′). All runs included 0 and 100% methylated human DNA as positive controls as well as a negative control. To confirm reproducibility every sample was tested two times and failed assays were repeated. Overall, intra-observer coefficient of variability between the two replicates of LINE-1 methylation measurements was 3.2% (SD = 3.0%). LINE-1 methylation levels were calculated as percentage of methylated cytosines over the sum of methylated and unmethylated cytosines, and reported for each CpG site as well as the average of the three CpG sites (GenBank Accession No. {"type":"entrez-nucleotide","attrs":{"text":"X58075","term_id":"34196","term_text":"X58075"}}X58075).