qPCR is used daily to validate array and sequencing results, but qPCR approaches have yet to dominate DNA methylation analysis the same way they have with other applications like gene expression analysis or genotyping. Things are a little bit more complicated when it comes to the epigenetic mods, but that hasn’t stopped hungry researchers from pillaging their toolboxes for fixes. Let’s take a look at some things that have come a long way recently with methylation qPCR
Bisulfite Sequencing: It’s Complicated
You’ve read about it. You’re probably using it. Bisulfite conversion remains a household name in most labs studying epigenetics, but extracting useful data can be difficult for many researchers. To start with, bisulfite can be really tough on DNA. Between degradation from the harsh conditions and purification losses, a hefty amount of DNA won’t ever make it past the conversion.
After conversion, other techniques are needed in order to get to the data you can use. Which means you’ve just signed up for several days worth of things like: PCR, gel extraction, ligation, cloning, and Sanger sequencing-most of which are made just a little trickier thanks to the C-to-U changes in converted DNA templates. For many researchers, the bisulfite-PCR-sequencing combo left a lot to be desired.
Beyond Bisulfite: First Generation Methyl Sensitive Restriction Enzyme (MSRE) qPCR
People have been coupling restriction digests with qPCR for years. It’s a nice alternative to the degrading lifestyle of high concentration sodium bisulfite, and provides a nice site-specific readout, but like a wacky ex, it has a few hangups that, well, we’ll just leave it at that. The big knock against MSRE is that it’s just not that convenient. Post-digestion, samples have had to be cleaned up before qPCR, which brings with it extra handling and manipulations, opening the door for sample loss and errors. Those extra steps also puts MSRE qPCR into the “low throughput” category, which for many in today’s high-throughput research environment is a deal breaker.
MSRE qPCR 2.0: One Step Methylation Analysis
Protocols rarely die nowadays, but are picked up, re-used, polished and re-deployed. Kind of like an ‘80’s hair-band riff that gets sampled by club DJs. We caught up with some scientists up at Zymo Research to hear more about how qPCR let them cut a remixed version of a high-throughput, quantitative DNA methylation analysis that has their R&D team hopping with excitement.
Like previous generations of MSRE qPCR, Zymo deploys a protocol that uses MSRE digests to give site-specific DNA methylation status and qPCR for precise quantitation of methylation levels, only now they’ve put them together in a way eliminates those old-school drawbacks.
The first thing you’ll notice about Zymo’s new OneStep qMethyl™ Kit is the simplified protocol. “The biggest breakthrough was the development of the buffer and enzyme system that allows for both MSRE digestion and qPCR within the same solution, making it a truly one-step process.” Explains Ron Leavitt from Zymo R&D. The Zymo team also threw in a sensitive dye to really dial in the qPCR reaction, and tackled data analysis with an easy and straight-forward web app that can be found online.
Digesting it All
What’s it all lead up to? Well a whole lot of hard work has transformed an outdated technique into something that’s way more useful. Here’s how the improvements ultimately pay off:
- One step: Now users can add all the solutions needed at one time to a 96-well plate where MSRE digestion and qPCR will all take place in the same well. No more 6-hour or overnight digestion followed by additional pipetting steps prior to qPCR like before. According to Leavitt, “What used to take 5-7 days worth of bench-work and waiting has been stripped down to only about 20 minutes of bench-work and 4 hours in a PCR instrument.”
- High throughput: Now you can look at several samples, or several loci, all on one plate. “OneStep qMethyl can analyze up to 44 samples in single-replicate, or 22 in duplicate, with room to spare for control DNAs and primers”, says Zymo R&D Scientist Lam Nguyen.
- Bisulfite free: No need to rehash the dark side of bisulfite, but we doubt many folks will miss it.
Flexible: From Basics to Biomarkers
Because of the features above, the upgraded protocol will work well in a variety of applications from basic research to biomarker screening. Want to screen CpG methylation status in several cell-types? No problem. The same is true for looking at multiple sites in a single cell type, or for double checking the methylation data you got from other methods before you move full steam into the next phase of a big project.
The one-step approach is also ideal for biomarker research. As Nguyen points out, “This technique allows a researcher to custom tailor the system for any epigenetic biomarker that needs to be quickly and accurately screened, and allows DNA methylation status to be quantitatively determined within hours.”
If you want to give DNAm qPCR a try in your next experiment and take advantage of this optimized method, check out the Zymo Research website to learn more about the OneStep qMethyl™ Kit.