LINE-1 methylation was carried out on the placental, maternal and cord blood DNA of all cases (Figure 1 ) in order to measure the global LINE-1 methylation pattern in the placenta to compare it with adult and cord blood, and to assess possible relationships with the birth-weight percentile.
LINE-1 methylation analyses were performed as previously described [41 (link)]. In brief, the PyroMark Q96 CpG LINE-1 kit (QIAGEN, Hilden, Germany) was used to amplify LINE-1 sequences from 20 ng of bisulfite-converted DNA and to quantify methylation levels at four CpG sites. Quantitative DNA methylation analyses were performed using pyrosequencing, using the Pyro Mark ID instrument (QIAGEN, Hilden, Germany), equipped with PSQ HS 96 System, and PyroGold SQA reagent kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Raw data were analyzed using the Q-CpG software v1.0.9 (Biotage AB, Sweden), which calculates the ratio of converted C’s (T’s) to unconverted C’s at each CpG, giving the percentage of methylation [50 (link)]. Reported methylation values are the mean between at least two independent PCR and pyrosequencing experiments.
LINE-1 methylation analyses were performed as previously described [41 (link)]. In brief, the PyroMark Q96 CpG LINE-1 kit (QIAGEN, Hilden, Germany) was used to amplify LINE-1 sequences from 20 ng of bisulfite-converted DNA and to quantify methylation levels at four CpG sites. Quantitative DNA methylation analyses were performed using pyrosequencing, using the Pyro Mark ID instrument (QIAGEN, Hilden, Germany), equipped with PSQ HS 96 System, and PyroGold SQA reagent kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Raw data were analyzed using the Q-CpG software v1.0.9 (Biotage AB, Sweden), which calculates the ratio of converted C’s (T’s) to unconverted C’s at each CpG, giving the percentage of methylation [50 (link)]. Reported methylation values are the mean between at least two independent PCR and pyrosequencing experiments.
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