Global DNA Methylation Analysis: The Best Assay You Haven’t Done Yet

A look at publications that leveraged ELISA to measure global 5-mC

MethylFlash Methylated DNA Quantification Kit (Colorimetric)

Over the past decade significant advances have been made in methylation profiling technology allowing for highly specific and accurate information about the epigenome of various species. Because the 5mC and 5hmC modifications are widespread with possibly different functions, further insight into their distribution is important.

Traditional methylation analysis methods such as mass spec, HPLC and TLC allow high accuracy but also require sophisticated equipment, are not high through-put and most importantly are expensive. Likewise more modern applications such as next generation sequencing come with a hefty price tag and result in massive amounts of data which initially may not be necessary. An ELISA-like method for studying global DNA methylation is a cost-effective, sensitive and accurate way of addressing several fundamental questions about DNA methylation and as an added bonus; it is also high-throughput.

Scientists at epigenetics company Epigentek commented that not only will a global DNA methylation approach establish a great foundation to build a study around, but “if multi-base resolution is your ultimate goal, starting out using a global detection assay is a sensible screening option which can save time and money by narrowing down your sample set.” Epigentek’s innovative technology has led them to have the most highly cited global DNA methylation assays with over 200 publications in top journals including Science, Nature Neuroscience, Cancer Research, and PLoS One.

Check out the featured citations below to see how researchers have modified a basic ELISA method to analyze global DNA methylation levels in a broad range of disciplines, as a fast, accurate and reliable alternative to other methods of DNA methylation profiling and maybe get an idea of how you can incorporate this application into your own study.


Autoimmune Disease:  In autoimmune disease, your immune system attacks healthy cells and tissue. Millions of people – mostly women – suffer from the chronic pain and illness associated with autoimmune ailments. The direct cause is still unknown but in addition to your genetic risk for developing one of these diseases, the epigenome may also play a role. The following studies have identified associations between DNA methylation and autoimmune disease.

Qin HH et. al. (May 2013). Associations between aberrant DNA methylation and transcript levels of DNMT1 and MBD2 in CD4+T cells from patients with systemic lupus erythematosus. Australas J Dermatol. 54(2):90-5.

Wiley KL et. al. (February 2013). Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus. J Clin Immunol. 33(2):342-8

Arakawa Y et. al. (November 2012). Association of polymorphisms in DNMT1, DNMT3A, DNMT3B, MTHFR and MTRR genes with global DNA methylation levels and prognosis of autoimmune thyroid disease. Clin Exp Immunol. 170(2):194-201.


Cancer and Cancer-Related Medical Conditions:  In 2013, 70% of epigenetic research was devoted to cancer/oncology as reported by Enal Razvi, Phd in Genetic Engineering and Biotechnology News; highlighting the major role of epigenetic related changes in cancer. Global DNA methylation profiling is an important first step in comparing disease states and is a method that can be efficiently used on large sample sets. The studies below have made significant findings using global DNA methylation analysis as part of their investigation.

Bujko M et. al. (May 2014). Repetitive genomic elements and overall DNA methylation changes in acute myeloid and childhood B-cell lymphoblastic leukemia patients. Int J Hematol.

Kang KA et. al. (April 2014). Epigenetic modification of Nrf2 in 5-fluorouracil-resistant colon cancer cells: involvement of TET-dependent DNA demethylation. Cell Death Dis. 5:e1183.

Yan H et. al. (April 2014). piRNA-823 contributes to tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma. Leukemia.

Nosho K et. al. (April 2014). Association of microRNA-31 with BRAF mutation, colorectal cancer survival and serrated pathway. Carcinogenesis. 35(4):776-83.

Kristensen DG et. al. (February 2014). Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis. Br J Cancer. 110(3):668-78.

Calvo X et. al. (May 2014). High levels of global DNA methylation are an independent adverse prognostic factor in a series of 90 patients with de novo myelodysplastic syndrome. Leuk Res.


Circadian Clock:  DNA methylation may also drive circadian clock plasticity.  Read more about how genome-wide methylation profiling revealed global alterations in promoter DNA methylation of the master clock tissue that regulates circadian behavior in mammals.

Azzi A et. al. (March 2014). Circadian behavior is light-reprogrammed by plastic DNA methylation. Nat Neurosci. 17(3):377-82.


Environmental Exposure:  The epigenetic alterations associated with short-term and long-term exposure to environmental chemicals and particulate matter is under intense study. Knowing the degree of global DNA methylation changes may give insight into future health risk related to these environmental factors.  These studies are hot off the presses and detail the relationship between DNA methylation changes and environmental factors.

Conti Ad et. al. (June 2014). Dose- and time-dependent epigenetic changes in the livers of fisher 344 rats exposed to furan. Toxicol Sci.139(2):371-80.

So MY et. al. (May 2014). Gene expression profile and toxic effects in human bronchial epithelial cells exposed to zearalenone. PLoS One. 9(5):e96404.

Miousse IR et. al. (May 2014). Epigenetic alterations induced by ambient particulate matter in mouse macrophages. Environ Mol Mutagen.55(5):428-35.

Tellez-Plaza M et. al. (April 2014). Association of Global DNA Methylation and Global DNA Hydroxymethylation with Metals and other Exposures in Human Blood DNA Samples. Environ Health Perspect.

Zhu JQ et. al. (February 2014). Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo. Arch Toxicol. 88(2):241-8.


Fertility, Prenatal Health and Postnatal Care:  The phrase “you are what your mother ate” has become commonly used to relay the idea that even before you are born, your epigenome can be altered to influence your future health and well-being. Take a look at how some researchers have investigated the effect of DNA methlylation on early development and its associations into adulthood.

Khalid O et. al. (May 2014). Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells. Stem Cell Res. 12(3):791-806.

Huang Y et. al. (April 2014). Maternal high folic acid supplement promotes glucose intolerance and insulin resistance in male mouse offspring fed a high-fat diet. Int J Mol Sci. 15(4):6298-313.

Weng X et. al. (February 2014). DNA methylation profiling in the thalamus and hippocampus of postnatal malnourished mice, including effects related to long-term potentiation. BMC Neurosci. 15:31.

SEE ALSO:   A New Study Shows MicroRNA-26a Targets DNA Demethylation Enzymes

Zhu JQ et. al. (February 2014). Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo. Arch Toxicol. 88(2):241-8.

Buscariollo DL et. al. (January 2014). Embryonic caffeine exposure acts via A1 adenosine receptors to alter adult cardiac function and DNA methylation in mice. PLoS One. 9(1):e87547.

Jenkins TG et. al. (October 2013). Paternal aging and associated intraindividual alterations of global sperm 5-methylcytosine and 5-hydroxymethylcytosine levels. Fertil Steril. 100(4):945-51.


Gene Expression and Regulation:  Epigenetic changes can both directly and indirectly affect gene expression and regulatory pathways. Here we highlight some recent studies which have investigated the effects of global DNA methylation changes in the regulation of specific gene elements and other cellular factors.

Basu A et. al. (April 2014). The CpG Island Encompassing the Promoter and First Exon of Human DNMT3L Gene Is a PcG/TrX Response Element (PRE). PLoS One. 9(4):e93561

Wang LJ et. al. (March 2014). Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoter and elevating genomic methylation in mice fed a high-fat diet. J Nutr Biochem. 25(3):329-36.

Guo F et. al. (February 2014). Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls. Reproduction. 147(2):241-52.

Ward A et. al. (January 2014). p53-Dependent and cell specific epigenetic regulation of the polo-like kinases under oxidative stress. PLoS One. 9(1):e87918.

Weiner AS et. al. (January 2014). Methylenetetrahydrofolate reductase C677T and methionine synthase A2756G polymorphisms influence on leukocyte genomic DNA methylation level. Gene. 533(1):168-72

Lou J et. al. (August 2013). Role of DNA methylation in cell cycle arrest induced by Cr (VI) in two cell lines. PLoS One.8(8):e71031.


Marine Biology:  Don’t be afraid to dive into the world of DNA methylation analysis.  Discover how scientists have applied a global DNA methylation approach to study how the epigenome affects marine growth and development.

Craig PM et. al. (February 2013). Methionine restriction affects the phenotypic and transcriptional response of rainbow trout (Oncorhynchus mykiss) to carbohydrate-enriched diets. Br J Nutr. 109(3):402-12.

Fang X et. al. (September 2013). Global and gene specific DNA methylation changes during zebrafish development. Comp Biochem Physiol B Biochem Mol Biol. 166(1):99-108.


Neuroscience: Neurological Disorders, Cognitive Function:  Several studies over recent years have also reported an association between epigenetic modifications and stress, anxiety, coping ability, long term memory and several neurological disorders. DNA methylation changes related to these outcomes could potentially serve as a predictive tool for clinical use. Below are a few studies that have studied DNA methylation as it relates to cognitive function and neurological disease.

Gaglio D et. al. (May 2014). Learning induced epigenetic modifications in the ventral striatum are necessary for long-term memory.Behav Brain Res. 265:61-8.

Wu X et. al. (April 2014). Epigenetic Signature of Chronic Cerebral Hypoperfusion and Beneficial Effects of S-adenosylmethionine in Rats. Mol Neurobiol.

Siuda D et. al. (April 2014). Social isolation-induced epigenetic changes in midbrain of adult mice. J Physiol Pharmacol. 65(2):247-55.

Bradley-Whitman MA et. al. (October 2013). Epigenetic changes in the progression of Alzheimer’s disease. Mech Ageing Dev.134(10):486-95.

Figueroa-Romero C et. al. (December 2012). Identification of epigenetically altered genes in sporadic amyotrophic lateral sclerosis. PLoS One. 7(12):e52672.

Chen H et. al. (March 2012). Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus. Restor Neurol Neurosci.30(3):237-45.


Plant Biology:  In addition to the intensely studied methylation changes in the human genome, DNA methylation also plays a significant role in plant gene expression and regulation. Furthermore the methylation profile in plants can be more complex and confusing than in humans. Luckily, using a global DNA methylation assay can make interpretation of plant DNA methylation data simple. Take a look at the reports below to see how.

Moricová P et. al. (March 2013). Changes of DNA methylation and hydroxymethylation in plant protoplast cultures. Acta Biochim Pol. 60(1):33-6.

Kim JE et. al. (December 2013). Characterization of non-CG genomic hypomethylation associated with gamma-ray-induced suppression of CMT3 transcription in Arabidopsis thaliana. Radiat Res. 180(6):638-48.

Testillano PS et. al. (September 2013). The 5-methyl-deoxy-cytidine (5mdC) localization to reveal in situ the dynamics of DNA methylation chromatin pattern in a variety of plant organ and tissue cells during development. Physiol Plant. 149(1):104-13.


Stem Cell Research:  The process of stem cell lineage commitment involves extrinsic and intrinsic signals that are strongly influenced by their microenvironment.  Furthermore, stem cell differentiation involves silencing of self-renewal genes and induction of a specific transcriptional program. It is not known how epigenetic modifications influences stem cell differentiation and commitment and what specific role these modifications may play. The articles below describe how DNA methylation and DNA hydroxymethylation may be involved in stem cell gene expression and differentiation.

Khalid O et. al. (May 2014). Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells. Stem Cell Res. 12(3):791-806.

Kim M et. al. (February 2014). Dynamic changes in DNA methylation and hydroxymethylation when hES cells undergo differentiation toward a neuronal lineage. Hum Mol Genet. 23(3):657-67.

Bose R et. al. (December 2012). Inherited effects of low-dose exposure to methylmercury in neural stem cells. Toxicol Sci.130(2):383-90.

Poloni A et. al. (May 2012). Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells. Stem Cells. 30(5):965-74.


Virology:  Here we have highlighted a few papers which describe the various ways in which DNA methylation was found to be either directly or indirectly associated with viral infection or the consequences of viral infection.

Wu J et. al. (March 2014). Kaposi’s sarcoma-associated herpesvirus (KSHV) vIL-6 promotes cell proliferation and migration by upregulating DNMT1 via STAT3 activation. PLoS One. 9(3):e93478.

Palacios JA et. al. (December 2012). Long-term nonprogressor and elite controller patients who control viremia have a higher percentage of methylation in their HIV-1 proviral promoters than aviremic patients receiving highly active antiretroviral therapy. J Virol. 86(23):13081-4.

Xu Q et. al. (February 2014). DNA methylation and regulation of the CD8A after duck hepatitis virus type 1 infection. PLoS One.9(2):e88023.

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