A cost-effective and scalable approach for DNA extraction from FFPE tissues.
Author(s): Geisenberger C, Chimal E, Jurmeister P, Klauschen F
Publication: Biol Methods Protoc, 2025, Vol. 10, Page bpaf003
PubMed ID: 39995602 PubMed Review Paper? No
Purpose of Paper
This paper compared the yield and purity of DNA, methylation profiles, and exome sequencing results of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue using the Maxwell RSC FFPE Plus DNA Purification Kit (Maxwell Kit) or a manual or automated version of the in-house manual method (HiTE) with either bead- or column-based purification. The effects of increasing the cross-link reversal incubation from 1 h to 16-24 h on DNA yield and purity were also examined. Additionally, the authors compared quantification of DNA with Qubit versus Nanodrop and investigated the potential influences of FFPE block storage duration and extraction method. The study used archival FFPE blocks of sarcoma (89 specimens) and used FFPE blocks of lung cancer specimens for methylation profiling (3 specimens) and exome sequencing (2 specimens).
Conclusion of Paper
DNA yields were much higher when DNA was isolated from FFPE specimens using the Maxwell Kit than the manual HiTE method with either bead- or column-based purification when the cross-link reversal incubation was 1 h; however, when cross-link reversal incubation was extended to 24 h, DNA yield increased significantly with the manual HiTE method, such that it was comparable to yields obtained with the Maxwell Kit. When HiTE extraction was automated for 96-well plates, the mean DNA yield was higher when purification was performed using beads rather than columns. DNA purity (A260/A280) using the manual HiTE method was not affected by the duration of the cross-link reversal incubation step or by the purification method (bead or column) following automated extraction; DNA purity was higher when column-based purification following manual extraction compared to when beads were used for purification. DNA concentrations determined by Qubit and NanoDrop were strongly correlated for FFPE specimens extracted using the manual or automated HiTE method but were only modestly correlated to one another when FFPE specimens that were extracted using the MaxWell Kit were also considered. As expected, absolute DNA concentrations were higher when samples were analyzed by NanoDrop than Qubit. The magnitude of the discrepancy in DNA yield between Qubit and NanoDrop was not affected by the storage duration of the FFPE block (1-12 years) or the DNA purification method; NanoDrop measurements could be corrected based on the geometric mean of the overestimation. The probe detection rate for the methylation arrays was >99.9% and the beta values had the expected bimodal distribution for all three specimens, regardless of the extraction method (automated HiTE with beads, automated HiTE with columns or MaxWell Kit). When the 5000 most variable probes were further analyzed, hierarchical clustering based on beta values clustered each of the three specimens by tissue source, not extraction method, and beta values for specimens were very strongly correlated between extraction methods (r=0.94-0.96). Finally, genome-wide copy-number plots were reproducible among the extraction methods examined. Exome sequencing had a mapping rate of >99%, a duplication rate of ~14% and an on-target rate of ~60% for both specimens using all three extraction methods (automated HiTE method with beads, automated HiTE method with columns, or MaxWell Kit). Plots of the cumulative normalized coverage for the exome regions by coverage were indistinguishable among the extraction methods, and the coefficient of variance for coverage uniformity between any two of the extraction methods (for either specimen) was low (≤0.01). The mutational signatures were also comparable among the extraction methods, with C-to -T transitions being the most frequent (~60%), which the authors state is a known artifact of FFPE processing.
Studies
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Study Purpose
This study compared the yield and purity of DNA, methylation profiles and exome sequencing results of DNA extracted from FFPE tissue using the Maxwell RSC FFPE Plus DNA Purification Kit (Maxwell Kit) or a manual or automated version of the in-house manual method (HiTE) with either bead- or column-based purification. The effects of increasing the cross-link reversal incubation from 1 h to 16-24 h on DNA yield and purity were also examined. Additionally, the authors compared quantification of DNA with Qubit versus Nanodrop and investigated the potential influences of FFPE block storage duration and extraction method. A total of 89 FFPE blocks containing sarcomas that were archived 1-7 years and 3 FFPE blocks containing lung cancer that were archived 9-12 years were used (no further details were provided). Regions with high tumor content were selected based on the H&E staining of 2 µM sections of each block. DNA was extracted from macroscopicly dissected regions with high tumor content using the Maxwell RSC FFPE Plus DNA Purification Kit (Maxwell Kit) and an in-house method termed HiTE that consisted of deparaffinization by incubation in mineral oil at 56°C for 15 min, lysis in a Tris-HCl (pH8.0) SDS buffer containing proteinase K at 56°C for 1 h, cross-link reversal by incubation for 1 or 16-24 h at 80°C, followed by bead-based (Ampure XP Beads) or column-based (DNAeasy Blood and Tissue Spin Column Kit) purification. DNA was quantified by Qubit HS DNA assay and by NanoDrop. For methylation analysis, DNA extracted from the three FFPE lung tumors was restored using the Illumina Infinium HD FFPE Restore Kit and bisulfite converted with the EpiTect Bisulfite Kit before analysis with Illumina Human Methylation Epic microarrays (v1 for Maxwell extracted and v2 for HiTE extracted). The data were normalized using single-sample normal-exponential out-of-band (Noob) normalization and analysis was performed using the software package tidyverse and conumee. Sequencing libraries were constructed using DNA from two of the lung cancer specimens (extracted with the automated HiTE method and purified with beads, the automated HiTE method using columns, and the MaxWell Kit) and the Exome 2.0 Kit from Twist Biosciences and were sequenced using an Illumina NovaSeq. Data was analyzed using R and Python scripts that included the Rsamtools, DescTools, vcfR, BSgenome and MutationalPatterns packages.
Summary of Findings:
DNA yields were much higher when DNA was isolated from FFPE specimens using the Maxwell Kit than the manual HiTE method regardless of purification method when the cross-link reversal incubation was 1 h; when cross-link reversal incubation was extended to 24 h, DNA yield increased significantly (P=2x10-5) when extraction was with the manual HiTE method, such that yields were comparable to those obtained with the Maxwell Kit. DNA concentrations measured by Qubit and NanoDrop were strongly correlated for specimens extracted using the manual HiTE method (r=0.95) but were only modestly correlated when specimens that were extracted using the MaxWell Kit were also considered (r=0.63). Nevertheless, as expected absolute concentrations were higher when samples were analyzed by NanoDrop than Qubit. DNA purity (A260/A280) of samples from the manual HiTE method was not affected by the duration of the cross-link reversal incubation; however, DNA purity was higher when DNA was purified using columns rather than beads. When the HiTE extraction method was automated for 96-well plates, mean DNA yield was higher when samples were purified with beads rather than columns (97.0 versus 68.6 ng/µL, P=0.023 by Qubit; 234.8 versus 157.6 ng/µL, P=0.009 by NanoDrop) but the purity was comparable between bead and column purification (1.84 and 1.9, respectively). Similar to manually processed specimens, DNA concentrations were strongly correlated between Qubit and Nanodrop measurements (r=0.85) but were ~2-fold higher using NanoDrop than Qubit. Importantly, the magnitude of the discrepancy in DNA yield between Qubit and NanoDrop was not affected by the storage duration of the FFPE block (1-12 years) or the purification method used (beads versus column); the NanoDrop measurement could be corrected based on the geometric mean of the overestimation. The probe detection rate for the methylation arrays was >99.9% and the beta values had the expected bimodal distribution for all three specimens, regardless of the extraction method (automated HiTE with beads, automated HiTE with columns or MaxWell Kit). When the 5000 most variable probes were further analyzed, hierarchical clustering based on beta value clustered each of the three specimens by tissue source, not extraction method, and beta values for specimens were very strongly correlated between extraction methods (r=0.94-0.96). Finally, the genome-wide copy-number plots were reproducible among the extraction methods examined. Exome sequencing had a mapping rate of >99%, a duplication rate of ~14% and an on-target rate of ~60% for both specimens using all three extraction methods (automated HiTE method with beads, automated HiTE method with columns or the MaxWell Kit). Further, plots of the cumulative normalized coverage for the exome regions by coverage were indistinguishable among the extraction methods evaluated, and the coefficient of variance for coverage uniformity was low (≤0.01) between any two of the extraction methods (for either specimen). The mutational signatures were also comparable among the extraction methods evaluated, with C-to -T transitions being the most frequent (~60%), which the authors state is a known artifact of FFPE processing.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA Spectrophotometry DNA DNA microarray DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1-12 years
Fluorometry Specific Technology platform Qubit
NanoDrop
Analyte Extraction and Purification Incubation duration/condition 1 h
16-24 h
Analyte Extraction and Purification Analyte isolation method Maxwell RSC FFPE Plus DNA Purification Kit
Automated HiTE
Manual HiTE
Analyte Extraction and Purification Analyte purification Magnetic beads-based
Silica column-based