title: Impact of tissue processing, archiving and enrichment techniques on DNA methylation yield in rectal carcinoma.
abstract: BACKGROUND
  Formalin fixation, duration of tissue storage and tissue enrichment techniques can affect DNA methylation yield but these effects have not been quantitatively measured. The aim is to investigate the relative impact of these conditions on DNA methylation in rectal cancer.
  
  
  METHODS
  10 rectal cancers with matched undissected fresh frozen tissues, laser capture microdissected (LCM) formalin-fixed paraffin-embedded (FFPE) tissues, manual macrodissected FFPE tissues, adjacent normal mucosa and stromal tissues were analysed for APC and LINE-1 methylation using bisulphite pyrosequencing.
  
  
  RESULTS
  FFPE cancer tissues, which had been stored for at least 4 years showed similar APC and LINE-1 methylation changes to matched fresh frozen cancer tissues. Laser capture microdissection did not increase the degree of methylation detected compared to manual macrodissection. Analysis of stromal tissues showed that they had undergone significant methylation changes compared to adjacent macroscopically normal mucosa, but not to the same extent as cancer tissues.
  
  
  CONCLUSIONS
  Reliable DNA methylation results can be obtained from FFPE rectal cancer tissues, which have been in long-term storage. Because only minor differences in methylation between macrodissected and LCM cancer tissues were found, our results do not support the routine use of LCM to enrich for cancer cells for DNA methylation studies.
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom. Electronic address: kjleong@gmail.com.
authors.forename: Kai Juen
authors.initials: KJ
authors.lastname: Leong
authors.forename: Jonathan
authors.initials: J
authors.lastname: James
authors.forename: Kaisheng
authors.initials: K
authors.lastname: Wen
authors.forename: Philippe
authors.initials: P
authors.lastname: Taniere
authors.forename: Dion G
authors.initials: DG
authors.lastname: Morton
authors.forename: Simon P
authors.initials: SP
authors.lastname: Bach
authors.forename: Glenn M
authors.initials: GM
authors.lastname: Matthews
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D019175:DNA Methylation; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000473:pathology*; D061346:Laser Capture Microdissection; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012004:Rectal Neoplasms / Q000235:genetics / Q000473:pathology*; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology*
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D005557:Formaldehyde
keywords: DNA methylation; Formalin-fixed paraffin-embedded tissues; Laser capture microdissection; Macrodissection; Rectal cancer; Stroma