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, 23 (1), 52-58

DNA Extraction From FFPE Tissue Samples - A Comparison of Three Procedures

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DNA Extraction From FFPE Tissue Samples - A Comparison of Three Procedures

Agnieszka K Sarnecka et al. Contemp Oncol (Pozn).

Abstract

Aim of the study: One of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA.

Material and methods: Slides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies).

Results: The results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method.

Conclusions: To our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

Keywords: Cobas; DNA extraction; DNA quality; FFPE tissue samples; Maxwell; Qiagen.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Scheme of the DNA isolation procedures
Fig. 2
Fig. 2
Quantitative analysis of DNA concentration in samples isolated from FFPE specimens. A) Spectrophotometric analysis with NanoDrop. B) Fluorimetric analysis with Qubit method. Results are presented as median and interquartile range (IQR) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig. 3
Fig. 3
Correlation of the concentration of DNA samples obtained with Cobas, Maxwell and QIAamp techniques. A) Spectrophotometric analysis with NanoDrop. B) Fluorimetric analysis with Qubit method. Correlations with Spearman coefficient in the range 0.4–0.7 were considered as moderate and with Spearman coefficient greater than 0.7 were regarded as strong. All correlations were statistically significant with p < 0.0001
Fig. 4
Fig. 4
Spectrophotometric assessment of extracted DNA quality expressed as an absorbance ratio of 260 nm to 280 nm (A260/A230). Median is shown with vertical solid lines. Error bars represent IQR. Dashed lines enclose high quality DNA samples with A260/A230 ratio falling within the range 1.8–2.0. Y-axis scale was narrowed to the range 1.0–3.0 to make the graph more transparent; therefore, four outlier points are not visible on the graph
Fig. 5
Fig. 5
Correlation of DNA concentration readings between spectrophotometric NanoDrop method and fluorimetric Qubit method

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References

    1. Ali N, Rampazzo RDCP, Costa ADiT, Krieger MA. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. Biomed Res Int. 2017;2017:9306564. - PMC - PubMed
    1. Donczo B, Guttman A. Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics. J Pharm Biomed Anal. 2018;155:125–134. - PubMed
    1. Williams C, Pontén F, Moberg C, Söderkvist P, Uhlén M, Pontén J, Sitbon G, Lundeberg J. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol. 1999;155:1467–1471. - PMC - PubMed
    1. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161:1961–1971. - PMC - PubMed
    1. Wong SQ, Li J, Tan AY, et al. Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genomics. 2014;7:23. - PMC - PubMed

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