Ensure High-Quality Nucleic Acids Analysis From FFPE Samples

How To Streamline FFPE Sample Preparation for High-Quality DNA and RNA AnalysisHow To Streamline FFPE Sample Preparation for High-Quality DNA and RNA Analysis www.covaris.com | 2 Introduction Next-generation sequencing (NGS) has revolutionized the field of genomics and is rapidly transforming clinical diagnosis and treatment across various disciplines, including oncology, hematology and immunology.1,2,3 Technological advances have increased the speed, accuracy and cost-effectiveness of NGS, providing clinicians with more comprehensive prognostic, diagnostic and therapeutic information. However, most tissue specimens in clinical practice are formalin-fixed, paraffin-embedded (FFPE), which are known to cause difficulties for NGS applications. FFPE is considered the gold standard for the long-term preservation of solid tissues and is extensively used in hospitals and clinics to preserve samples for retrospective studies, experimental research and ongoing clinical diagnostics.4,5,6 Despite its widespread use, extracting nucleic acids from FFPE samples for NGS analysis is still challenging due to the variability in the quality of these specimens. The fixation process can lead to DNA fragmentation, cross-linking, and unwanted chemical modifications, compromising the integrity and yield of nucleic acids required for high-quality sequencing.7,8 Additionally, the age of the FFPE sample, variations in fixation conditions and the presence of inhibitors further impact the quality of the extracted genetic material and, consequently, the outcomes of downstream genomic analyses.9,10,11 Although there are several methods available for extracting nucleic acids from FFPE specimens, they are often labor-intensive and can involve harmful processes to the sample, such as toxic chemicals or exposure to high heat. Additionally, these methods risk incomplete deparaffinization, which can render the tissue inaccessible to downstream enzymatic processes. Therefore, there is a pressing need to streamline FFPE extraction methods to obtain high-quality nucleic acids efficiently and safely, ensuring the integrity of samples for NGS and other molecular applications. The challenges of working with FFPE samples Researchers have long known that the formalin fixation process has substantial downstream effects on genomic applications, particularly for the detection of false-positive mutations.12 Although formalin does not physically degrade nucleic acids, it produces DNAand RNA–protein cross-links which reduce both the amplifiable quantity and length of nucleic acids.13,14 This complicates analytical processes like PCR amplification and NGS, where intact and high-quality nucleic acids are essential for accurate results. The extraction workflow involves several steps including deparaffinization, tissue lysis, reverse-crosslinking and DNA purification.15 Deparaffinization, the removal of paraffin to make nucleic acids accessible, is particularly important and also affects the amplifiability of extracted nucleic acids.16 A central component of almost all deparaffinization protocols is xylene, which poses multiple challenges (Figure 1):How To Streamline FFPE Sample Preparation for High-Quality DNA and RNA Analysis www.covaris.com | 3 • Time-consuming: Deparaffinization requires multiple washing steps with organic solvents.17 • Laborious: These processes require a lot of manual work and are difficult to automate. 17 • Requires working in a fume hood: Due to the hazardous nature of solvents like xylene, all procedures must be conducted under a fume hood to ensure safety, limiting flexibility and scalability.18 • Chemical waste disposal: The use of xylene and ethanol necessitates proper disposal of chemical waste, adding to the environmental and operational burden.19 • Contamination risk: Multiple handling steps increase the risk of sample contamination. • Hazardous: Xylene is both toxic and flammable, posing significant health and safety risks to laboratory personnel. 19 • Tissue loss: Multiple washes can lead to the loss of tissue material, further compromising sample integrity.20 • Poor yields of low-quality DNA: The harsh conditions and extensive handling often result in low yields of fragmented and degraded DNA, unsuitable for highfidelity genetic analysis. Overall, the workflow is complex, difficult to automate and challenging to scale up, limiting its efficiency and effectiveness. This complexity poses a significant barrier to the widespread use of FFPE samples in largescale genomic studies and clinical applications. How can you overcome these obstacles? As the demand for FFPE tissues continues to grow, there is an increasing need for safer substitutes for xylene that reduce costs, save time and produce a standardized, reliable workflow for large-scale studies.21 Several alternatives have been explored, including various types of oils, hot water and commercially available solutions like Histo-Clear®, Sub-X®, Bioclear® and UltraClear™.17,22,23,24,25 While many of these solutions are less hazardous than xylene, many still rely on complex and time-consuming protocols. Figure 1. Traditional methodologies for nucleic extraction from FFPE samples rely on organic solvents for deparaffinization, resulting in a time-consuming and laborious workflow. Requires chemical Requires a fume hood waste disposal Time-consuming Laborious Multiple washes can Damaged DNA lead to tissue loss Serious health hazard Risk of contaminationHow To Streamline FFPE Sample Preparation for High-Quality DNA and RNA Analysis www.covaris.com | 4 One promising solution to these challenges is the use of Adaptive Focused Acoustics® (AFA®), delivered on Covaris Focused-ultrasonicators. This technique uses focused ultrasound to process samples through highly controlled bursts of high-frequency acoustic energy (Figure 2). By precisely controlling the creation and collapse of millions of cavitation bubbles within the sample vessel, AFA generates sub-micron shear forces that effectively remove paraffin from FFPE samples. This method is rapid, non-contact and temperaturecontrolled, ensuring efficient and reproducible sample processing without the hazards associated with traditional chemicals like xylene.26,27 Already the gold standard for shearing DNA and RNA for NGS applications, AFA technology has also been successfully applied to RNA sequencing, ELISAs, liposome preparation methods, preparation of nanosuspensions and FFPE-based proteomics.28,29,30,31,32,33 As a well-established technology within biomolecular applications, AFA enables the rapid and reliable extraction of high-quality nucleic acids from FFPE samples for high-throughput applications. Focused-ultrasonicator Transducer Water Bath Covaris Consumable Focal Zone AFA Figure 2. Adaptive Focused Acoustics (AFA) technology employs highly controlled bursts of focused high-frequency acoustic energy to process FFPE samples. These acoustic processes are performed in an isothermal, non-contact environment. How to get the most out of your FFPE samples When working with FFPE samples, it’s crucial to maximize the quality and quantity of extracted nucleic acids. AFA technology provides several advantages compared to traditional techniques: 1. No toxic organic solvents required Since traditional FFPE sample processing often relies on hazardous organic solvents for paraffin removal, protocols need to include specialized waste disposal and fume hoods. In contrast, AFA technology uses aqueous buffers, streamlining the workflow and reducing associated costs and safety concerns. 2. Simultaneous paraffin removal and tissue rehydration AFA simultaneously emulsifies paraffin into an aqueous buffer and rehydrates the tissue in 5 minutes or less, thereby enabling more efficient proteinase K digestion and de-crosslinking kinetics (Figure 3). This innovative approach minimizes the inherent variability between samples and the degradation of nucleic acids for downstream analysis. Before AFA Treatment After AFA Treatment Figure 3. AFA technology can be used to simultaneously remove paraffin and rehydrate tissue in 5 minutes or less. 3. Simultaneous extraction of NGS-quality DNA and RNA from FFPE tissue samples While truXTRAC FFPE kits are available for extracting either DNA or RNA separately, AFA technology also enables the efficient and simultaneous extraction of both DNA and RNA from the same FFPE sample. This allows researchers to reliably compare genomic and transcriptomic data (Figure 4). This is particularly important when working with tumor tissues, which often contain a heterogeneous mix of healthy and malignantHow To Streamline FFPE Sample Preparation for High-Quality DNA and RNA Analysis www.covaris.com | 5 Reverse Crosslinks DNA Purification Reverse Crosslinks Centrifuge DNA from pellet Analysis Paraffin Removal and Tissue Re-hydration DNA Fragmentation RNA from supernatant Tissue Digestion RNA Purification Figure 4. Using AFA technology, both RNA and DNA can be extracted from the same sample. Figure 5. The truXTRAC FFPE kits, using AFA technology, yield similar or greater amounts of both DNA and RNA compared to competitor kits. Additionally, AFA technology produces superior DV200 scores for RNA, outperforming traditional methods. Brain Breast Tumor Tissue Type Yield (µg) Colon 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Kit Competitor truXTRAC-Auto truXTRAC-Manual DNA Brain Breast Tumor Tissue Type Yield (µg) Colon 5 4 3 2 1 0 Kit Competitor truXTRAC-Auto truXTRAC-Manual RNA RNA DV 200 Score Comparison Traditional Method 6/79 samples had DV200 > 20% Covaris 90/90 samples had DV200 > 20% DV200How To Streamline FFPE Sample Preparation for High-Quality DNA and RNA Analysis www.covaris.com | 6 cells. Extracting DNA and RNA from different sections can generate data from different cellular populations, leading to potential discrepancies. Simultaneous extraction prevents this issue, ensuring reliable data and minimizing sample wastage. 4. High-quality DNA and RNA results Combined with AFA technology, truXTRAC® FFPE kits provide robust and reliable assays designed for every lab and user, ensuring consistent results each time. These kits have been demonstrated to achieve similar or higher yields and better amplifiable nucleic acids compared to other commercial kits tailored for FFPE samples (Figure 5). 27 One key metric for RNA quality is the DV200 score, which measures the percentage of RNA fragments greater than 200 nucleotides in size. Given that FFPE samples often contain degraded RNA, the DV200 score is a reliable predictor of successful library preparation and sequencing results. Notably, truXTRAC FFPE kits have achieved significantly higher DV200 scores for RNA than traditional techniques, indicating superior RNA quality and better outcomes for downstream applications (Figure 5). 5. One complete automatable workflow from deparaffinization to shearing For higher throughput and even greater efficiency, truXTRAC FFPE SMART Solutions provide a scalable approach that encompasses the entire sample preparation process, from deparaffinization to DNA shearing. These automation-friendly kits streamline sample preparation and consistently deliver robust nucleic acid yields, critical for maximizing biomarker detection in applications such as comprehensive genomic profiling.34 The R230 Focused-ultrasonicator can be fully integrated with various liquid handling platforms, enabling the automation of the entire process on a single instrument. This integration reduces variability and provides the versatility and scalability required for high-throughput processing, making it an ideal solution for modern laboratory workflows. Conclusion FFPE tissue samples are set to play a major role in the future of personalized healthcare, providing a wealth of information for studying disease development, progression and treatment efficacy. However, current methods for extracting nucleic acids from FFPE samples are time-consuming and often result in low yields of degraded nucleic acids, which are unsuitable for NGS analysis. With the introduction of scalable and automationfriendly technologies like truXTRAC FFPE SMART Solutions, researchers can finally unlock the vast archives of FFPE samples for comprehensive analysis. AFA technology streamlines the extraction process, significantly reducing the time required while ensuring high yields of intact, high-quality nucleic acids. This advancement not only enhances the feasibility of largescale studies using FFPE samples but also improves the reliability of the data obtained, ensuring more accurate and reproducible results. 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