It’s human nature to get enthralled by the latest, shiny new technology on the market, but sometimes a more established approach is just as effective. We recently connected with our friends at Zymo Research who point out that for DNA methylation analysis, researchers may not always need all of NGS’s bells and whistles, especially when you can often get the info you seek with a simple, tried and true DNA methylation ELISA. Check out how some Zymo customers are using old-school technology to get quick and cost-effective DNA methylation data.
Going Global with DNA Methylation Analysis
With the recent advances in high-throughput sequencing platforms, there has been a tremendous rush to apply that technology to DNA methylation analysis, but it’s not always necessary to map 5mC and/or 5hmC down to the nucleotide level or even regional context.
Many researchers have opted to develop global DNA methylation screens based on familiar and reliable methods such as ELISAs and LC-MS for a cost-effective and rapid means to monitor whole-genome DNA methylation without a sequence-specific context.
Let’s take a look at some of the more prominent research settings where DNA methylation screening is playing a key role.
Comparative DNA Methylation Profiling
It is highly valuable for researchers to have the ability to quickly and efficiently assess differential DNA methylation between two groups. There are multiple scenarios where global DNA methylation screens help investigators to zero-in only where it matters most.
Differential Drug Response
ELISA assays, like the 5-mC DNA ELISA and the Quest 5-hmC DNA ELISA Kits from Zymo Research, have made it relatively simple to see the effects that certain drug treatments have on cell populations. Here are some examples where our customers were able to monitor the effects of compound exposures with DNA methylation screens:
- Scientists from the university of Turin(Riganti, et.al., 2013) observed that 5-mC levels in glioblastoma stem cells rose nearly 25% when treated with temozolomide, an oral chemotherapy agent, which chemosensitized the stem cells.
- Investigators at Loma Linda University School of Medicine (Paradis et. al., 2014) found that exposure to endothelin-1 (ET-1), which is expressed during hypoxia, increased 5-mC in fetal cardiomyocytes, but 5-Aza-2-deoxycytidiine could block that effect.
Did You Know? DNA Methylation ELISAs Can Efficiently Pre-Screen Samples for NGS
High-throughput sequencing based studies require a big investment in time, capital, equipment and resources. So, it makes sense to be judicious about what you choose to throw into each sequencer run. With a quick ELISA DNA methylation assay, you can decide exactly what deserves some more in-depth, locus specific attention, and which samples are better left out…all at a fraction of the cost and effort. It’s hard to argue with efficiency.
5-mC and 5-hmC ELISAs have also been deployed to track DNA methylation alterations in response to environmental stimuli such as diet and air quality. These recent publications detail how DNA methylation screens measure the effects of environmental factors:
- These University of Louisville authors (Kalani et. al., 2014) set out to study the neuroprotective properties of Folic Acid. They observed no 5-mC changes in mice that were fed Folic acid in their diets, but did notice that when cystathionine-β-synthase (a hyperhomocysteinemia related enzyme) was included, global 5-mC levels increased in mouse brain tissue.
- Researchers at the National Institutes of Health (Cheng et.al., 2014) investigated the role of epigenetics in asthma and allergic responses. The group showed that exposure to house-dust mites (HDM) lowered global 5-mC in mouse lungs, but also increased the percentage of 5-hmC measured in comparison to control mice treated with saline.
Differential Disease States and Tissue Types
Rapid assays, like ELISAs, are extremely helpful in initial screens for potential DNA methylation shifts between tissue types or disease conditions. The studies below used this approach to identify differential DNA methylation levels between healthy and diseased cells, in order to conduct more locus specific experiments later:
- A research team at Johns Hopkins University (Tellez-Plaza et. al., 2014) determined the 5-mC and 5-hmC levels in cardiovascular disease-free men.
- The authors then went on to correlate their data with and abnormal metabolic conditions or low levels of atherosclerosis to generate an initial dataset for future studies.Scientists from the University of Louisville (Chaturvedi et. al., 2014) studied 5-mC levels in cardiomyocytes that were undergoing hyperhomocysteinemia (HHcy), which is associated with neurovascular diseases via aberrant DNA methylation and histone acetylation. They observed that HHcy cardiomyocytes indeed, had elevated 5-mC levels compared to wild type cells.
- Investigators at the Universities of Bergen and Oslo, (Bjanesoy et. al. 2014) while looking into Autoimmune Addison’s Disease (AAD) quantified the 5-mC levels of CD4+ T cells. The team showed that AAD patients had much lower 5-hmC levels than healthy subjects.
Stem Cell Monitoring
Another emerging application of DNA methylation screens is the monitoring of cultured stem cells. Several recent reports have demonstrated that stem cell properties can shift significantly over time, depending on variables like culture conditions and passage number, greatly impacting their experimental behaviour and clinical utility. (Schellenberg et. al., 2011 and Redaelli et. al., 2012)
Researchers have determined that keeping an eye on epigenetic factors in stem cells, like DNA methylation, with the help of relatively fast and simple screening assays can indicate the state of a stem cell population. This can either be accomplished in a couple of ways:
- Global DNA methylation screens including ELISAs and LC-MS
- Or with a more focused, panel approach such as the one utilized in the OneStep qMethyl™ Human Pluripotent Stem Cell Panel from Zymo Research
Here are a few examples of DNA methylation being used to assess stem cells:
- Investigators from the University of Aachen (Schellenberg et. al., 2011) delved into the effects of long-term culture in human mesenchymal stem cells (MSC). They compared DNA methylation profiles and noted a substantial difference between MSCs derived from adipose tissue and bone marrow. All MSCs had highly consistent senescence-associated DNA methylation modifications that also correlated with specific histone marks including H3K9me3, H3K27me3 and EZH2. Their observations led the team to conclude that replicative senescence is epigenetically controlled.
- Later on, the same scientific team at the University of Aachen (Schellenberg et. al., 2014) developed an Epigenetic-Senescence-Signature to predict cellular aging in mesenchymal stem cells (MSCs) by analysis of DNA methylation at six CpG sites (associated with the genes GRM7, CASR, PRAMEF2, SELP, CASP14 and KRTAP13-3). The group used this project as proof of principle, and intends to further validate this approach for use as a quality control of therapeutic cell products.
- Authors of this report from the University of Milan-Bicocca (Redaelli et. al., 2012) found that the large-scale expansion of bone marrow mesenchymal stem cells (BM-MSCs) required for use in a therapeutic context can initiate replicative senescence. This was linked to a significant reduction in BM-MSC 5-mC levels during long-term culture.