Quantifying LINE-1 DNA Methylation

Genomic DNA was extracted from the tumor and modified with sodium bisulfite using an EpiTect Bisulfite Kit (Qiagen). PCR and subsequent pyrosequencing for LINE-1 were performed as previously described by Ogino et al., using the PyroMark Kit (Qiagen) [24 (link)–27 (link)]. This assay amplifies a region of the LINE-1 element (position 305–331 in accession no. X58075), which includes four CpG sites. The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as a percentage (i.e., 0–100%). The average of the relative amounts of C in the four CpG sites was used as the overall LINE-1 methylation level in a given tumor. We validated our LINE-1 methylation pyrosequencing assay in the published literature [28 (link)].

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Murata A., Baba Y., Ishimoto T., Miyake K., Kosumi K., Harada K., Kurashige J., Iwagami S., Sakamoto Y., Miyamoto Y., Yoshida N., Yamamoto M., Oda S., Watanabe M., Nakao M, & Baba H. (2015). TET family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma. Oncotarget, 6(27), 23372-23382.

Publication 2015

EpiTect Bisulfite Kit PyroMark Kit

Assay Bisulfite Genomic Line 1 Methylation Sodium bisulfite Tumor

Corresponding Organization : Kumamoto University

Other organizations : National Hospital Organization Kyushu Cancer Center, Japanese Foundation For Cancer Research

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Variable analysis

independent variables

Sodium bisulfite modification of genomic DNA from the tumor

dependent variables

LINE-1 methylation level in the tumor
Relative amount of C (cytosine) to the sum of C and T (thymine) at each of the four CpG sites in the LINE-1 element

control variables

PyroMark Kit (Qiagen) used for PCR and pyrosequencing of LINE-1
Assay amplifying a region of the LINE-1 element (position 305-331 in accession no. X58075), which includes four CpG sites

controls

The authors validated their LINE-1 methylation pyrosequencing assay in the published literature [28 (link)].

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