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Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples

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Pathology & Oncology Research

Abstract

We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from “recent”, collected within 6 months, and “archived”, collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18μg DNA; Q: 3.00 ± 4.04μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91μg DNA; Q: 11.51 ± 7.50μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

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Acknowledgments

This study was supported by the National Research, Development and Innovation Office (KMR-12-1-2012-0216 grant) and Hungarian Scientific Research Fund (OTKA-103244 and OTKA-K111743 grants). The authors would like to gratefully acknowledge the support and assistance from Roche Diagnostics GmbH. We would like to thank Gabriella Kónyáné Farkas (Semmelweis University) for her technical assistance in the study.

Conflict of Interest

The Authors declare no conflict of interest.

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    Authors and Affiliations

    1. 2nd Department of Internal Medicine, Semmelweis University, Szentkirályi utca 46, 1088, Budapest, Hungary

      Alexandra Kalmár, Bálint Péterfia, Árpád V. Patai, Andrea Schöller, Zsolt Tulassay & Béla Molnár

    2. Molecular Medicine Research Unit, Hungarian Academy of Sciences, Szentkirályi utca 46, 1088, Budapest, Hungary

      Alexandra Kalmár, Bálint Péterfia, Barnabás Wichmann, Zsolt Tulassay & Béla Molnár

    3. 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085, Budapest, Hungary

      Péter Hollósi & Tibor Krenács

    4. Tumor Progression Research Group, Hungarian Academy of Sciences, Roosevelt tér 9, 1051, Budapest, Hungary

      Péter Hollósi

    5. Department of Physics of Complex Systems, Eötvös Loránd University, Pázmány Péter sétány 1/A, 1117, Budapest, Hungary

      András Bodor

    6. 2nd Department of Surgery, Semmelweis University, Kútvölgyi út 4, 1125, Budapest, Hungary

      Andrea Schöller

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    1. Alexandra Kalmár
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    Correspondence to Alexandra Kalmár.

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    Alexandra Kalmár and Bálint Péterfia contributed equally to this work.

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    Kalmár, A., Péterfia, B., Hollósi, P. et al. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples. Pathol. Oncol. Res. 21, 1149–1156 (2015). https://doi.org/10.1007/s12253-015-9945-4

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    • DOI: https://doi.org/10.1007/s12253-015-9945-4

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