Researchers and clinicians want to leverage formalin-fixed paraffin-embedded (FFPE) tissues for genomic, transcriptomic, epigenetic, and proteomic analyses. But the nucleic acid quality and integrity in FFPE specimens are affected by numerous factors including fixative pH, duration of tissue fixation, age and storage condition of tissue blocks, and the extraction method.1
“Clinical samples, especially oncology biopsies, are typically FFPE tissues. It is extremely difficult to accurately extract functional DNA and RNA from these samples,” says Roman Rodriguez, Covaris’ Director of Marketing. “At Covaris, we optimized and streamlined sample prep methodologies for paraffin-embedded, crossed-linked samples through partnerships. Our technology removes paraffin, rehydrates the tissue, and extracts biomolecules much more efficiently than conventional unoptimized methods using organic solvents. We can now mitigate the inherent variability related to each sample and/or prevent damage to the biomolecules that may lead to inconsistent analysis quality.”
Already the gold standard for shearing DNA and RNA for genome sequencing applications, the patented Adaptive Focused Acoustics (AFA™) technology is setting new standards in FFPE-related DNA, RNA, chromatin, and protein extractions.
Unlike conventional probe and bath sonicators, AFA is based on highly controlled, focused, high-frequency acoustic energy. High-density pressure fluctuations trigger dissolved gasses to form millions of microscopic cavitation bubbles, generating shear forces. The short, ~3mm AFA wavelength enables controlled focusing within a precise zone that results in effective, isothermal, and reproducible sample preparation.
Minimal energy input eliminates adverse excess-energy effects, such as damaging heat, variability, and sample over-processing. The Covaris technology simultaneously emulsifies paraffin from FFPE into an aqueous buffer and rehydrates the tissue; thereby, enabling more efficient proteinase K digestion and de-crosslinking kinetics. This results in increased yields of nucleic acids and minimization of the degradation of nucleic acids.
Collaboration with HudsonAlpha: Improved DV200
The HudsonAlpha Institute for Biotechnology has a mission of conducting genomics-based research to improve human health and implementing genomic medicine.
Often, FFPE samples are degraded and may, therefore, contain RNA fragments smaller than the optimal size range for efficient target-capture and library preparation. To compensate, DV200, a metric that measures the percentage of RNA fragments >200nt in size, is often used as a reliable predictor of successful library preparation and downstream sequencing results.
According to Shawn Levy, director of the Genomic Services Laboratory at HudsonAlpha, the truXTRAC™ extraction showed significant improvement in the percentage of RNA recovered larger than 200nt in length (DV200 scores) compared to other methodologies, where only 20% of FFPE samples had DV200 scores of greater than 20%. With the Covaris truXTRAC method, recent analysis of over 400 FFPE samples resulted in 80% of samples with DV200 greater than 20% with a mean DV200 of 45%, and co-extracted DNA showed similar yields and quality compared to other methods.
Covaris truXTRAC FFPE kits allow manual or automated extraction of nucleic acids. The kits facilitate next-generation sequencing (NGS) data complexity and enhanced detection of recurrent gene fusions.1
Collaboration with OmniSeq®: Automation for clinical sample prep
OmniSeq, a clinical laboratory, utilizes NGS technologies to assist in drug development and to guide oncology treatment decision-making.
An extraction process that reliably produces sufficient DNA yield and fragment sizes from precious FFPE samples is a necessity for any molecular pathology laboratory. In another comparison, OmniSeq demonstrated that the truXTRAC kit provided an efficient system for generating high-quality DNA samples from lung cancer specimens that failed extraction by a silica-based method.2
DNA requirements for genomic testing were reduced 25–50% without loss of assay performance or analytic sensitivity. The combination of improved yield and fragment size suggest the feasibility of using even smaller biopsies for advanced diagnostic testing.
The Covaris LE220R-plus Focused-ultrasonicator was also integrated with a liquid-handling robotics platform to scale and standardize a clinical FFPE extraction workflow; thus, allowing better clinical analysis and more robust outcomes.
“By using the Covaris truXTRAC DNA and RNA kits, OmniSeq testing requires less tissue and results in fewer specimen failures while performing comprehensive genomic profiling on solid tumors,” said Jeff Conroy, CSO of OmniSeq.
Similar cDNA and DNA library preparation yields and quality were also observed when comparing manual and automated workflows. Downstream sequencing performance results were significantly above their threshold metric levels, providing confidence in the analysis. Similar copy numbers of somatic variants also were detected.3
AFA can be fine-tuned to process samples for many new applications, from low-power gentle solution mixing and protein extraction, to higher-power needs for DNA fragmentation, microbial lysis, nanoliposome formation, and nanoparticle creation.
Covaris is innovating with a new on-deck Focused-ultrasonicator to be integrated on your liquid handlers. It uses 96 or 384-well Covaris plates (soon 1536) and is amenable to high-throughput clinical automation. The newly designed Revolution R230 is available for pre-launch testing.
Contact: Roman Rodriguez, firstname.lastname@example.org
1. Kresse et al. (2018) Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples. PLoS ONE 13(5): e0197456
2. Conroy et al. (2015) DNA Extraction of Lung Cancer Samples for Advanced Diagnostic Testing, 16th World Congress on Lung Cancer poster
3. Glenn et al. (2018) Novel Automated Co-extraction of High-quality DNA and RNA from a Single FFPE Sample, AMP poster