Abstract
In DNA methylation, methyl groups are covalently bound to CpG dinucleotides. However, the assumption that methyl groups are not lost during routine DNA extraction has not been empirically tested. To avoid nonbiological associations in DNA methylation studies, it is essential to account for potential batch effect bias in the assessment of this epigenetic mechanism. Our purpose was to determine if the DNA isolation method is an independent source of variability in methylation status. We quantified Global DNA Methylation (GDM) by luminometric methylation assay (LUMA), comparing the results from 3 different DNA isolation methods. In the controlled analysis (n = 9), GDM differed slightly for the same individual depending on extraction method. In the population analysis (n = 580) there were significant differences in GDM between the 3 DNA isolation methods (medians, 78.1%, 76.5% and 75.1%; p<0.001). A systematic review of published data from LUMA GDM studies that specify DNA extraction methods is concordant with our findings. DNA isolation method is a source of GDM variability measured with LUMA. To avoid possible bias, the method used should be reported and taken into account in future DNA methylation studies.
Highlights
Epigenetic mechanisms regulate high-order DNA structure and gene expression without affecting the DNA nucleotide sequence
DNA samples extracted by method 1 (Autopure LS), had a median Global DNA Methylation (GDM) of 77.2% with an interquartile range (IQR) of 75.5–77.8; method 2 (Puregen TM), 76.0% (IQR 74.5–76.8), and method 3 (Chemagic), 76.2% (IQR 75.5–76.5)
No statistically significant differences were found between the three methods due to the small sample size, substantial variation in values was observed, considering that all the three methods were tested on samples from the same individuals with blood extraction done at the same time (Figure 1)
Summary
Epigenetic mechanisms regulate high-order DNA structure and gene expression without affecting the DNA nucleotide sequence. Three main epigenetic mechanisms of gene regulation have been described: DNA methylation, histone modification, and noncoding RNA. Methylation, the most widely studied epigenetic mechanism, is a genomic DNA mark resulting from a covalent bond of a methyl group to the 5-carbon position of cytosine, generally in a 59-CpG39 context. This dinucleotide is rare in the genome (,1%) and tends to form clusters known as CpG islands, which are usually unmethylated and located in gene promoter regions. DNA methylation occurs at CpG island shores, in the gene body, and in repetitive elements [1,2,3,4]. Changes in DNA methylation contribute to inter-individual phenotypic variation and are associated with cancer development and other complex diseases [5,6]
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