title: Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.
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 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg 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.
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Szentkirályi utca 46, 1088, Budapest, Hungary, alexandra.kalmar@gmail.com.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kalmár
authors.forename: Bálint
authors.initials: B
authors.lastname: Péterfia
authors.forename: Péter
authors.initials: P
authors.lastname: Hollósi
authors.forename: Barnabás
authors.initials: B
authors.lastname: Wichmann
authors.forename: András
authors.initials: A
authors.lastname: Bodor
authors.forename: Árpád V
authors.initials: ÁV
authors.lastname: Patai
authors.forename: Andrea
authors.initials: A
authors.lastname: Schöller
authors.forename: Tibor
authors.initials: T
authors.lastname: Krenács
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D010232:Paraffin / Q000737:chemistry*; D016612:Paraffin Embedding / Q000379:methods; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D013447:Sulfites / Q000737:chemistry*
chemical_list: D013447:Sulfites; D005557:Formaldehyde; D010232:Paraffin; D004247:DNA; C042345:hydrogen sulfite