EZ-96 DNA Methylation-Gold™ Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Genomic DNA was isolated from buffy coat of cord blood and from placental tissue using the QIAamp DNA mini kit (Qiagen, Inc., Venlo, the Netherlands). We performed DNA methylation analysis by highly quantitative bisulfite-PCR pyrosequencing. Bisulfite conversions were performed using 1 µg of extracted genomic DNA with the EZ-96 DNA methylation Gold kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. The final elution volume was 40 µl, using M-elution buffer. We interrogated CpG sites within promoter regions (SIRT1, TP53, PPARG, PPARGC1A, TFAM) or other specific regions (subtelomere: D4Z4; mitochondrial genome: MT-RNR1, D-loop) of targets on the mitochondrial-telomere axis. We combined data from the literature and data derived from the UCSC Genome Browser (http://genome.ucsc.edu,assemblyGRCh37/hg19), such as transcription factor binding sites, DNase I hypersensitive sites and histone modifications to identify regions with potentially methylated CpG sites. The MethPrimer program (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) was used for PCR primer design. The assays for D4Z4, MT-RNR1 and D-loop have been previously described [31, 32]. Detailed information regarding primer sequences and genomic regions is given in Supplementary file 1, Table 1. Methylation levels of the mitochondrial regions MT-RNR1 and D-loop showed a strong correlation in placental tissue (n = 81, r = 0.85, p < 0.0001) and cord blood (n = 82, r = 0.68, p < 0.0001).

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Manufacturer protocol

Download the product protocol from Zymo Research for EZ-96 DNA Methylation-Gold™ Kit below.

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