Quantifying Age-Related DNA Methylation

DNA isolation and telomere length measurements in the EpiPath were reported previously in Elwenspoek et al. (manuscript under review, Journal of Immunology). Methylation levels were measured at age-related CpGs in aspartoacylase (ASPA) (cg02228185), integrin alpha 2b (ITGA2B) (cg25809905), and PDE4CA (cg17861230) according to (22 (link)). In brief, unmethylated cytosine residues in each DNA sample were converted to uracil with a bisulfite treatment (EpiTect Bisulfite Kit, Qiagen, Venlo, Netherlands) and regions of interest were amplified with PCR (PyroMark PCR Kit, Qiagen) in the bisulfite-modified DNA according to the manufacturer’s protocols. PCR products were pyrosequenced on a Pyromark ID with Pyrogold reagents (Biotage, Uppsala, Sweden) and methylation levels were analyzed with Pyro Q-CpG SW (Biotage). A sample of pooled DNA was run in each batch as internal control, which was used to calculate relative methylation levels. These relative methylation levels were used for all further analyses.

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Elwenspoek M.M., Sias K., Hengesch X., Schaan V.K., Leenen F.A., Adams P., Mériaux S.B., Schmitz S., Bonnemberger F., Ewen A., Schächinger H., Vögele C., Muller C.P, & Turner J.D. (2017). T Cell Immunosenescence after Early Life Adversity: Association with Cytomegalovirus Infection. Frontiers in Immunology, 8, 1263.

Publication 2017

EpiTect Bisulfite Kit PyroMark PCR Kit Pyromark ID Pyrogold reagents

A dna Aspartoacylase Bisulfite Cytosine Integrin Isolation Methylation Telomere Uracil

Corresponding Organization : Luxembourg Institute of Health

Other organizations : Universität Trier, University of Luxembourg

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Variable analysis

independent variables

Methylation levels at age-related CpGs in ASPA (cg02228185), ITGA2B (cg25809905), and PDE4CA (cg17861230)

dependent variables

Relative methylation levels

control variables

Sample of pooled DNA run in each batch as internal control

controls

Positive control: Sample of pooled DNA run in each batch
Negative control: Not explicitly mentioned

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