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Expert Insight: 5-hmC Analysis Methods

August 5, 2010 Last year’s discovery of 5-hydroxymethylcytosine (5-hmC) caused some epigenetics researchers to suffer a crisis of confidence, provoking many hours of soul-searching questions such as “are my methylation data really accurate?” and “what if what I thought was gene silencing is really activation?” Although current estimates place 5-hmC levels at only a fraction of the 5-mC content in most cells, it could still be enough to foul up your data interpretation, especially if 5-hmC’s function ends up being antithetical to 5-mC’s as some researchers have proposed.

Sizing Up the 6th Base

Since enzymatic and bisulfite-based methods can’t distinguish methylcytosine (5-mC) from 5-hmC, product developers have had to develop alternate ways to detect the modified base. Some of these new methods enable locus-specific analysis, while others provide a global snapshot of 5-hmC presence. Let’s take a look...

Locus-Specific 5-hmC Analysis

5-hmC Immunoprecipitation Approaches

If you’ve ever done MeDIP, the principle here is the same, except that you use one of the recently developed antibodies that specifically target 5-hmC. First you enrich for 5-hmC-marked DNA by immunoprecipitation and then run it through your downstream weapon of choice (e.g. sequencing, qPCR, arrays) to quantify the presence of 5-hmC in any given sequence.

Active Motif was quick to launch two antibodies that target 5-hmC: a rabbit serum version and a purified IgG version. The purified version is ideal for applications in which you need to know exactly how much IgG you’re using. According to Jim Bone, strategic marketing manager with Active Motif, the company also has a 5-hmC monoclonal antibody in the works.

Diagenode currently sells the only monoclonal, a rat monoclonal antibody, that targets 5-hmC. “Diagenode offers the most specific antibody for the study of 5-hmC,” claims Didier Allaer, CEO of Diagenode. The company also has an hMeDIP kit that includes the monoclonal 5-hmC antibody and materials for a magnetic bead-based assay, which can be completed in only 24 hours. For quality control, the kit supplies hydroxymethylated, methylated, and unmethylated DNA standards.

The hMeDIP approach is a definitely a step forward in being able to profile 5-hmC patterns, but selective enrichment is still going to be challenging when 5-hmC and 5-mC are in close proximity.

5-hmC Global Analysis

Some researchers may still need convincing that 5-hmC is relevant to their work. So far, the modification seems to be most prevalent in ES cells. Say you’re working with fibroblasts – should you really care? Luckily, you can resolve this nagging question by taking a quick look at global 5-hmC levels in your cells with these approaches.

5-hmC HPLC- and MS-based Approaches

HPLC and LC/MS offer precise 5-hmC quantitation, but where do you start? Zymo Research offers products to prepare samples for these techniques. DNA Degradase™ and DNA Degradase Plus™ enzyme cocktails degrade DNA into single nucleotides or nucleosides, respectively. Then, you can use TLC, HPLC (DNA Degradase™), or LC/MS (DNA Degradase Plus™) to quantitate global 5-mC and 5-hmC. “The strength of these products is that the reactions are simple to set up and quick to run,” says James Yen, scientist at Zymo Research. “DNA samples can be completely processed and ready for analysis within 1 hour.”

To help with calibrating the detection systems, Zymo offers a set of three DNA standards with identical sequences, except for the modification status of cytosines: all unmodified (C), all methylated (5mC), or all hydroxymethylated (5hmC). “When defining a calibration curve for MS, known ratios of the standards are mixed,” explains Yen. “Then, the methylation read outs of the experimental DNA can be quantitated against the curve to define precise global methylation/hydroxymethylation levels.”

5-hmC Colorimetric Plate Assay

Another option for assessing global levels of 5-hmC is Epigentek’s MethylFlash Hydroxymethylated DNA Quantification Kit. With this kit, you bind your genomic DNA to wells in a microplate and then spectrophotometrically detect the 5-hmC fraction with capture and detection antibodies. “Any lab that’s ever done ELISA will find this kit a breeze to use,” says Adam Li, chief scientific officer at Epigentek. “Because the kit is so much more practical and affordable than MS or HPLC, now any lab with a microplate spectrophotometer can do 5-hmC studies on a routine basis.” The kit is fast (less than 4 hours), amenable to high-throughput analyses, specific (“There’s no cross-reactivity to 5-mC and unmethylated C,” Li says), and sensitive (20-ng detection limit of input DNA).

Li notes that Epigentek is currently developing a version of the MethylFlash kit for use with a fluorescence-based reader. “The fluorescent version will have a detection limit as low as 5 pg of 5-hmC DNA, which will allow its use with samples only available in small amounts such as laser capture microdissection samples and embryonic cells,” he says.

Early Findings

When developing and testing these new products, company researchers were bound to make exciting discoveries about the prevalence and possible role of 5-hmC. They were kind enough to share with us some fascinating unpublished data that you’ll only see here. Yen reveals that scientists at Zymo research detected 5-hmC in zebrafish embryo DNA, suggesting that this mysterious epigenetic mark is also conserved in lower vertebrates.

At Epigentek, scientists using MethylFlash technology discovered that 5-hmC distribution in various human tissues and cells can vary by greater than 50-fold, with brain, liver, kidney, and colon tissues having the highest 5-hmC contents (>0.3%), lung, heart, placenta, and breast tissues much lower (<0.1%), and cell lines the lowest of all (<0.05%). “Most important and interesting is that 5-hmC in cancerous tissues is significantly decreased – 1/3 to 1/4 of the corresponding normal tissues,” says Li.

Stay Tuned

In the short amount of time since 5-hmC’s discovery, these companies have delivered an impressive array of options for sizing up the 6th base. But this is only the beginning—the company reps assure us that they have lots of exciting products in the works, so stay tuned. In the very near future, you may have to attribute those sleepless nights to some other research boogeyman.

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