Quantitative DNA Methylation Analysis

Purified genomic DNA from tissue samples was used for global long interspersed nucleotide element-1 (LINE-1) and miR-137 promoter methylation analyses. The procedure was in detail described in our previous reports^{36 (link),40 (link)}. Briefly, we applied Cells-to-CpG Bisulfite Conversion Kit (Life Technologies, Carlsbad, CA) for bisulphite modification, thereafter the standard PCR with biotin-labelled primers and eventually the pyrosequencing on PyroMark Q96 ID (Qiagen) using PyroMark Gold Q96 reagents (Qiagen). The mean methylation level of analysed CpG motifs was used for quantitative methylation analysis.

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Boehm E.T., Thon C., Kupcinskas J., Steponaitiene R., Skieceviciene J., Canbay A., Malfertheiner P, & Link A. (2020). Fusobacterium nucleatum is associated with worse prognosis in Lauren’s diffuse type gastric cancer patients. Scientific Reports, 10, 16240.

Publication 2020

Cells-to-CpG Bisulfite Conversion Kit PyroMark Q96 ID PyroMark Gold Q96 reagents

Biotin Bisulphite Cells Genomic Gold Long interspersed nucleotide element 1 Methylation Mir 137 Primers Tissue

Corresponding Organization : Otto-von-Guericke University Magdeburg

Other organizations : Lithuanian University of Health Sciences

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Variable analysis

independent variables

Purified genomic DNA from tissue samples

dependent variables

Global long interspersed nucleotide element-1 (LINE-1) methylation
MiR-137 promoter methylation

control variables

Bisulfite modification using Cells-to-CpG Bisulfite Conversion Kit (Life Technologies, Carlsbad, CA)
Standard PCR with biotin-labelled primers
Pyrosequencing on PyroMark Q96 ID (Qiagen) using PyroMark Gold Q96 reagents (Qiagen)
Mean methylation level of analyzed CpG motifs

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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