A couple of years ago, researchers keen to analyze 5-hmC had a couple of options, but those early approaches couldn’t quite satisfy all of their increasing demands. Fast-forward a bit and it’s a very different story. Today there’s a plethora of options, each with its own strengths and weaknesses. As a result, one of the biggest challenges researchers currently face in 5-hmC analysis arises long before experiments are conducted: choosing the right tool for the job.
A Brief History of 5-hmC Analysis
Skirmantas Kriaucionis and Nathaniel Heintz kicked off the 5-hmC analysis wave with a Science publication back in 2009, after they noticed an odd spot on their TLC plates while analyzing some Purkinje DNA samples.
In this guide…
- We introduce various methods for global, locus-specific, and single-nucleotide 5-hmC analysis
- Review the strengths of each of the approaches
- Discuss the technical expertise required for each approach
- Summarize the material costs associated with each approach
They quickly deployed both HPLC and MS approaches to confirm that this odd spot was 5-hmC. These TLC, HPLC, and MS-based approaches were accurate methods and they were instrumental in catapulting 5-hmC into the spotlight, but their ability to help researchers get to the bottom of exactly what role 5-hmC played and where it was located was limited.
In this same publication, Heintz and Kriaucionis suggested what later groups would confirm; bisulfite conversion (at least in isolation), and other popular 5-mC analysis methods would not detect 5-hmC at all, or wouldn’t be able to discern it from 5-mC. Opportunistic academic and commercial scientists dove into their toolkits, and within months a variety of new approaches surfaced.