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Abstract
Introduction: Archived FFPE tissue specimens are a valuable resource for advancing cancer-related health disparities research. Retrospectively collected tissues help minimize follow-up time and prevent delays in research progress. While kits for DNA purification from FFPE are readily available, challenges such as limited tissue in FFPE blocks lead to insufficient DNA yield, and suboptimal quality persists. In this article, we have optimized and standardized methods for purifying DNA with high yield and integrity, making it highly suitable for generating DNA libraries and conducting high-throughput sequencing, thereby enhancing the reliability and reproducibility of cancer genomics studies.
Materials and Methods: We employed two Qiagen kits: the QIAamp DNA FFPE Tissue Kit and the QIAamp DNA FFPE Advanced Kit. To address challenges related to limited tissue in FFPE blocks, low yields, and poor quality for metagenomic sequencing library preparation, we systematically modified the protocol to maximize both yield and quality, even from the scarce scrolls available. The quantity and quality of the extracted DNA were assessed using a NanoDrop 2000 spectrophotometer and a Qubit dsDNA Broad-Range assay. DNA integrity was evaluated with a Bioanalyzer and TapeStation. Libraries were then prepared using Illumina kits.
Results: Optimizing the DNA extraction protocol led to a significant improvement in both yield and quality from FFPE tissues. Compared to the manufacturer's protocols, DNA yields increased by 82%, as confirmed by NanoDrop 2000 and Qubit assays. Additionally, DNA integrity, evaluated using a Bioanalyzer and TapeStation, demonstrated preservation of higher molecular weight fragments, with the DNA Integrity Number (DIN) improving from 3.2 to 7.2. These enhancements not only boost the overall quality of the extracted DNA but also ensure its suitability for high-throughput sequencing, metagenomic studies, and other advanced genomic applications.
Conclusion: Both the Qiagen DNA FFPE Kit and the Qiagen DNA FFPE Advanced Kit are ideal for purification of DNA from FFPE tissues. By significantly improving DNA yield and integrity, our optimized protocol enables the reliable generation of DNA libraries and supports high-throughput sequencing applications. This advancement enhances the utility of archived FFPE specimens in cancer-related health disparities research, paving the way for more reproducible and impactful genomic studies.
Recommended Citation
Singh, Shweta; Perez, Astrid; Vidaurri, Sierra; Dhasmana, Anupam; Dhasmana, Swati; Yallapu, Murali Mohan; Chauhan, Subhash C.; and Khan, Sheema, "Maximizing DNA Yield and Integrity from Limited FFPE Tissue: A Breakthrough Approach for Advancing Cancer Genomics" (2025). Research Symposium. 150.
https://scholarworks.utrgv.edu/somrs/2025/posters/150
Included in
Maximizing DNA Yield and Integrity from Limited FFPE Tissue: A Breakthrough Approach for Advancing Cancer Genomics
Introduction: Archived FFPE tissue specimens are a valuable resource for advancing cancer-related health disparities research. Retrospectively collected tissues help minimize follow-up time and prevent delays in research progress. While kits for DNA purification from FFPE are readily available, challenges such as limited tissue in FFPE blocks lead to insufficient DNA yield, and suboptimal quality persists. In this article, we have optimized and standardized methods for purifying DNA with high yield and integrity, making it highly suitable for generating DNA libraries and conducting high-throughput sequencing, thereby enhancing the reliability and reproducibility of cancer genomics studies.
Materials and Methods: We employed two Qiagen kits: the QIAamp DNA FFPE Tissue Kit and the QIAamp DNA FFPE Advanced Kit. To address challenges related to limited tissue in FFPE blocks, low yields, and poor quality for metagenomic sequencing library preparation, we systematically modified the protocol to maximize both yield and quality, even from the scarce scrolls available. The quantity and quality of the extracted DNA were assessed using a NanoDrop 2000 spectrophotometer and a Qubit dsDNA Broad-Range assay. DNA integrity was evaluated with a Bioanalyzer and TapeStation. Libraries were then prepared using Illumina kits.
Results: Optimizing the DNA extraction protocol led to a significant improvement in both yield and quality from FFPE tissues. Compared to the manufacturer's protocols, DNA yields increased by 82%, as confirmed by NanoDrop 2000 and Qubit assays. Additionally, DNA integrity, evaluated using a Bioanalyzer and TapeStation, demonstrated preservation of higher molecular weight fragments, with the DNA Integrity Number (DIN) improving from 3.2 to 7.2. These enhancements not only boost the overall quality of the extracted DNA but also ensure its suitability for high-throughput sequencing, metagenomic studies, and other advanced genomic applications.
Conclusion: Both the Qiagen DNA FFPE Kit and the Qiagen DNA FFPE Advanced Kit are ideal for purification of DNA from FFPE tissues. By significantly improving DNA yield and integrity, our optimized protocol enables the reliable generation of DNA libraries and supports high-throughput sequencing applications. This advancement enhances the utility of archived FFPE specimens in cancer-related health disparities research, paving the way for more reproducible and impactful genomic studies.
