Find statistically relevant biomarkers in tissue that has been stored in paraffin for decades.

Measuring epigenetic markers in paraffin embedded (FFPE) blocks is challenging. Fixation extensively damages and shreds DNA. The nucleotides of these DNA fragments also become altered by processes such as depurination and de-amination.

The EpiRestore™ service restores heavily degraded DNA so it can be analyzed using Illumina’s MethylationEPIC micro-array. Moreover, our modified hybridization protocol allows you to start with as little as 200 ng of DNA. You can harvest a wealth of data of previously unattainable patient cohorts.

DNA repair service

Our scientists have developed a proprietary DNA repair service that allows you to measure methylated and/or hydroxymethylated residues. This advanced method interrogates over 850,000 loci throughout the genome, selected by epigenetic experts. It delivers robust and highly reproducible results despite the very poor condition of the starting material.

Quality checks

We perform quality checks throughout the whole procedure, to ensure that only good quality results will be reported. We deliver all micro-array quality specs already loaded into the intuitive open-source software package MethylAID (van Iterson, M. et al. Bioinformatics 2014). You can immediately assess the quality of the data by scrolling through the graphs. Each describe a quality aspect: sample-dependent as well as sample-independent controls such as call-rates, hybridization controls and bisulfite conversion controls.

What extent of DNA degradation can EpiRestore™ handle?

Four samples were selected that are representative for the range of samples that researchers encounter after DNA isolation from FFPE blocks. Additional types of DNA damage of the nucleotides -such as de-purination and oxidation- cannot be visualized by gel-electrophoresis.

• Tumors from a cohort dating from 1988, split in half and either fresh frozen or preserved as FFPE blocks
• Average fragment size between 400 and 700 basepairs
• EpiRestore™ service restored the FFPE DNA
• Performed the fine-tuned protocol of the Illumina’s MethylationEPIC array to accommodate samples with low input material.

Each probe or CpG locus is represented multiple times on the array. The average of the fluorescent signals of each of these 850,000 loci (average beta-value or Avβ of each probe) is visualized in the figure as a black dot.

Results:

• EpiRestore™ greatly increases the concordance of severely degraded FFPE samples with their cognate Fresh Frozen aliquot
• The more heavily damaged DNA, the greater the improvement of the results
• EpiRestore™ significantly improves the biological relevance of the data in all samples

Experiment:

• Cohort of samples, extracted from FFPE
• Input concentration of all samples standardized on 200 ng or less
• Average fragment size below 400 bp (Undamaged DNA: >20,000 bp)
• EpiRestore™ service: DNA restoration followed by the adjusted MethylationEPIC protocol
• qPCR analysis to quantitatively and qualitatively measure the DNA before starting the micro-array experiment.

Results:

• Positive control consisting of good quality DNA: ~100% of the loci detected
• The higher the Ct value on the x-axis, the lower the quality of the DNA
• Our qPCR method shows a tight correlation between quality and call rates, the detected number of CpGs.
• Even the most heavily degraded samples (fragment size peak at ~200 bp) show a call rate of 70%: the dataset of this sample contains the methylation status of >765,000 methylation sites (p>0.01).

For freshly isolated DNA, we routinely detect call rates of above 99%. We can guarantee to detect 95% of the loci with a p-value of p≤0.01). With our fine-tuned protocols we can start this procedure with 100 ng input or even less, in contrast to the 250 ng input recommended by Illumina.  Contact technical support to inquire.
The results obtained by EpiRestore™ are depending on the quality of the DNA. We guarantee that at least 90% of the CpGs are detected at p<0.01 for a minimum of 80% of the samples. Thus, at least ~765,000 CpG loci that show differential methylation patterns in e.g. health and disease, are called with a high level of statistical certainty.
This guarantee is valid for input amounts of at least 200 ng, enabling you to select only the regions of interest from the paraffin blocks.

Finding Biomarkers with consortium partners

Biomarkers are characteristics by which a disease can be identified or monitored. They reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. GenomeScan partners now with many (academic) institutes to find diagnostic and predictive biomarkers for cancer and other diseases, based on epigenetic markers on the DNA. We participate in multiple consortia. Visit our research collaborations page to learn about how to successfully apply for funding together with us.