Genome-wide DNA methylation analysis of archival formalin-fixed paraffin-embedded tissue using the Illumina Infinium HumanMethylation27 BeadChip.

Author(s): Thirlwell C, Eymard M, Feber A, Teschendorff A, Pearce K, Lechner M, Widschwendter M, Beck S

Publication: Methods, 2010, Vol. 52, Page 248-54

PubMed ID: 20434562 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if incorporation of a ligation step prior to WGA would allow comparable determination of methylation status from FFPE to that from frozen ovarian tumor specimens. The ability to apply the protocol to FFPE specimens representing 6 tissue types stored for up to 10 years was also investigated. DNA was extracted from frozen and histoclear deparaffinized FFPE specimens with QIAamp and an additional an overnight proteinase K digestion for FFPE specimens.

   Summary of Findings:

   DNA from FFPE specimens that was ligated with Repli-g, prior to WGA, had a mean fragment size of 5 kb, but without ligation, the FFPE DNA failed to amplify. Ligated DNA from FFPE specimens showed a correlation of 0.96 in methylation status to unamplified frozen specimens, but without ligation, the correlation to frozen specimens dropped to 0.74. Further, the correlation between technical replicates was higher among ligated FFPE DNA than unligated FFPE DNA. A similar number of CpG sites were successfully interrogated using ligated DNA from FFPE specimens stored < 1 month or 6 months and in unamplified frozen specimens. Finally, ligation prior to WGA allowed for interrogation of CpG sites in endometrium, liver, prostate, pancreas, lymph node and neuroendocrine FFPE specimens stored 3-10 years.

   Biospecimens

   • Tissue - Ovary
   • Tissue - Liver
   • Tissue - Endometrium
   • Tissue - Prostate
   • Tissue - Pancreas
   • Tissue - Lymph Node
   • Tissue - Brain

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Whole genome amplification

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh) Frozen
   Biospecimen Acquisition	Biospecimen location	Ovary Endometrium Liver Prostate Pancreas Lymph node Neuroendocrine
   Storage	Storage duration	<1 month 6 months 3 years 6 years 8 years 10 years
   Whole genome amplification Specific	Template modification	Ligated Unligated

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