Sooner or later researchers studying DNA methylation turn it up a notch and get into some difficult sample scenarios, where the interesting data hides tucked away. We caught up with the R&D scientists at Zymo Research to hear more about some of the challenges that researchers face when stepping up into some of these sample scenarios:
- Profiling Blood or serum samples
- High-Throughput Bisulfite Analysis
- Formalin fixed and paraffin embedded (FFPE) samples
DNA Methylation Profiling in Blood
Blood contains a variety of DNA, RNA, and protein biomarkers that could be invaluable in prognostic and diagnostic settings if they can be reliably monitored. Clinical researchers have been chomping at the bit to pin down DNA methylation biomarkers in this non-invasive sample for years, but unlike more homogenous sample types, blood can be a very tricky specimen to assay DNA methylation.
The heterogeneous composition of blood introduces a major challenge in reliably assaying any biomarker. This is made even more difficult when trying to consistently detect a modification like DNA methylation residing on a subset of available sequence. To make matters worse, the amount of available DNA in blood samples can be minute.
Bisulfite conversion provides researchers the ability to detection differential DNA methylation at any nucleotide, making it a very attractive approach. That said, the process can be harsh on samples, leading to significant sample degradation and loss. This is problematic in any sample scenario, but it is even more so with clinical samples like blood given how little assayable material is available at the start of the process.
“When working with blood, it’s critical to use the most sensitive bisulfite conversion approach possible, since every last bit of material is precious,” shared Seth Ruga, a Sr. Researcher with Zymo Research.
In many cases, researchers might try to use milder conversion conditions, but this can lead to false DNA methylation status.
“We use a direct conversion protocol that is designed to convert blood samples directly, with no need for purification beforehand. This helps us minimize sample loss from small samples of DNA during treatment and clean-up,” according to Ruga. “Ultimately this has helped us amplify bisulfite converted templates from as few as 10 cells or 50 pg DNA.”
High Throughput DNA Methylation Profiling
Bisulfite sequencing remains the most prominent way to perform targeted DNA methylation profiling. Researchers seeking to profile a few loci across large number of samples, or validate large numbers of candidate loci from genome-wide DNA methylation studies, often find themselves needing to run hundreds, if not thousands of bisulfite sequencing reactions.
Although sequencing has been optimized for high throughput analysis for years, bisulfite conversion hasn’t. Many researchers struggle with the transition to high throughput bisulfite-based DNA methylation analysis. The multi-channel pipette, although useful, can still introduce plenty of errors and variation in sample preparation.
“When our customers are transitioning into higher numbers of samples, we urge them to consider using a magnetic bead based clean-up for high-throughput methylation analysis.” This way, desulphonation and clean-up of your bisulfite converted DNA can be performed while bound to the beads, stated Ruga. “Bead-based purification approaches are typically compatible with a wide variety of automation platforms as well, which will greatly enhance the reproducibility of the study, not to mention reduce labor dramatically.”
DNA Methylation Profiling in FFPE Samples
Much of the world’s tumors samples have undergone formalin fixation and paraffin embedding (FFPE). That’s great news for preserving valuable samples with immense amounts of historical data for cancer studies, but bad news for most analytical methods to study these tumors.
The major issue with FFPE samples is they’ve undergone harsh treatment during the preservation process. The same formalin reagent that preserves them causes all kinds of crosslinking between proteins and nucleic acids. On top of the fixation process, most of these samples subsequently undergo paraffin embedding to further stabilize the samples. The whole process is rough on samples, leading to fragmented, tough to use nucleic acids.
As if the FFPE process wasn’t harsh enough, it also requires researchers to reverse much of the preservation process before it’s possible to analyze the samples. Removing the paraffin treatment and reversing crosslinks can be just as damaging as the initial preservation process.
Given the damaged nature of methylated DNA in FFPE samples, it is extremely important to minimize any further damage or sample loss with the bisulfite conversion process.
“Whenever you’re working with such minute amounts of viable DNA, you want to do everything possible to eliminate any steps that might cause additional template damage or loss,” according to Ruga. “Clean-up steps and precipitations, no matter how efficient, can lead to sample loss.”
“Much like with blood or LCM samples, a direct conversion system, where you can perform bisulfite conversion without prior purification, will dramatically improve sensitivity in DNA methylation studies with FFPE samples,” added Ruga.