At first pass it may seem like all bisulfite conversion kits are essentially the same – and for the most part, that’s true. But according to a recently published evaluation, the conversion is all in the details, and if those are overlooked it can have a big impact on the results.
Fortunately, researchers from the University of Freiburg set out to compare bisulfite conversion kits from prominent manufacturers using pyrosequencing as their measuring stick, and in the process found some nice little hacks to get the best out your own bisulfite reactions. The team created assays for four genes (ALUSX, IGF2, IL6ST and ST3GAL2) with varying degrees of DNA methylation, and then ran each kit through its paces. While all of the kits worked well most of the time, there were certain conditions where adjustments were needed to get optimal results.
The Freiburg crew found two main ways to tweak incubation protocols to get the consistency they wanted: Incubation Time (Short vs. Long) and Incubation Type (Fixed Temp vs. Temp. Cycling).
Bisulfite Conversion Time is On Your Side
“Faster is better” has been the motto for the latest bisulfite kits on the market. However the researchers found that when variable conversion efficiencies did pop up, it could often be addressed by extending the reaction time. They noticed that a 16 hr protocol performed better than either the 8hr or 3hr versions. So, next time your boss is breathing down your neck about speeding up a bisulfite protocol, tell them that good things come to those who wait.
Temperature Cycling Keeps the Bisulfite Wheels Turning
It’s usually best to just keep things moving; especially when it comes to bisulfite reaction temperatures. The scientists noticed that sticking with a fixed temperature held back the conversion reaction, while using a thermocycling strategy proved to be much more effective. Go ahead, spice up that fixed temperature program a little bit.
Bisulfite Kit Breakdown
The authors chose five of the most popular bisulfite conversion kits out there to compete in their matchup. Based on manufacturer recommended protocols, they all performed as expected with consistent results, except for a just a couple of situations.
While the three Zymo kits worked well across the board, the Qiagen and Active Motif entries required some tinkering when it came to the IL6ST (low methylation) target. In both cases, switching to a ’cycling 16 h’ protocol fixed things up nicely.
- Zymo Research
- EZ DNA Methylation: Performed well in all assays.
- EZ DNA Methylation-Gold : Performed well in all assays.
- EZ DNA Methylation-Lightning: Performed well in all assays.
- EpiTect+: Performed well in 3 of 4 assays; Variable results in low methylation assay fixed by cycling 16h protocol.
- Active Motif
- MethylDetector: Performed well in 3 of 4 assays; Variable results in low methylation assay fixed by cycling 16h protocol.
According to the report, any of the kits that were looked at will get the job done nicely with the right set up. The authors also noted that use of a long thermocycling approach is a great way to standardize conversion efficiencies across kits from different suppliers.
Get more bisulfite pointers at Medical Epigenetics, April 2014