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

Alexandra Kalmár, Bálint Péterfia, Péter Hollósi, Barnabás Wichmann, András Bodor, Árpád V. Patai, Andrea Schöller, Tibor Krenács, Zsolt Tulassay, Béla Molnár

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1149-1156
Number of pages8
JournalPathology and Oncology Research
Volume21
Issue number4
DOIs
Publication statusPublished - Sep 28 2015

Keywords

  • Colorectal cancer
  • DNA methylation
  • Genomic DNA isolation
  • Methylation-sensitive high-resolution melting analysis
  • Pyrosequencing

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Oncology
  • Cancer Research

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