Development and validation of an optimized, bead-based dual nucleic acid extraction method for high-yield genomic profiling of scarce FFPE tumor specimens

Formalin-fixed, paraffin-embedded (FFPE) tumor tissues are crucial for retrospective oncology studies, but recovering high-quality nucleic acids (NA) from these samples-especially scarce core needle biopsies (CNBs)-is challenged by chemical crosslinking, fragmentation, and low input volume, often compromising next-generation sequencing (NGS) success. To meet the demand for reliable NGS from limited FFPE material, we developed and validated a Modified Proprietary Dual Extraction Method specifically optimized for low-input and highly degraded FFPE tissues, including CNBs. This novel, bead-based chemistry utilizes a Xylene-free deparaffinization step, followed by Covaris UltraSonication to achieve optimized lysis and de-crosslinking conditions. The method focused on refining key technical parameters, namely lysis buffer concentration and de-crosslinking time/temperature, and was benchmarked against our Internal Standard and a commercial column-based standard (Qiagen AllPrep FFPE Dual Kit). FFPE samples across multiple tumor types (kidney, head and neck, bladder, lung) and varying tissue inputs (0.5 mm³ to 4.0 mm³) were tested, including a cohort of known low-quality (LowQ) FFPE samples. We evaluated DNA/RNA yield, integrity (DIN/RIN), and reproducibility across all three methods. The Modified Method demonstrated significantly superior performance: DNA Yield and Recovery: The protocol delivered a dramatic increase in usable DNA yield, with 98.83% of samples meeting the minimum 100 ng NGS threshold, compared to only 73.50% using the commercial kit. DNA Quality: The Modified Method achieved higher DNA yields without compromising DNA integrity and notably improved the quality metrics of challenging LowQ FFPE specimens. RNA Quality: RNA integrity also significantly improved over the AllPrep Kit, enhancing the suitability of the resulting RNA for complex downstream applications like RNA sequencing (RNA-seq) workflows. The protocol exhibited high reproducibility with minimal operator or lot-to-lot variability. This Modified Dual Extraction Method, leveraging optimized bead-based chemistry and sonication, enables superior, high-quality, and reproducible recovery of nucleic acids from the most challenging FFPE tumor specimens. Its validated performance over conventional protocols is critical for advancing reliable genomic profiling in cancer research and precision oncology using archived or limited tissue specimens.