title: The tissue is the issue: improved methylome analysis from paraffin-embedded tissues by application of the HOPE technique.
abstract: Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.
authors.affiliation: 1] Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany [2] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Marwitz
authors.affiliation: Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Kolarova
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] LungenClinic Grosshansdorf, Grosshansdorf, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Reck
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] LungenClinic Grosshansdorf, Grosshansdorf, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Reinmuth
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] LungenClinic Grosshansdorf, Grosshansdorf, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kugler
authors.affiliation: Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Schädlich
authors.affiliation: Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Haake
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] Medical Clinic, Research Center Borstel, Borstel, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Zabel
authors.affiliation: 1] Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany [2] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany.
authors.forename: Ekkehard
authors.initials: E
authors.lastname: Vollmer
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Reiner
authors.initials: R
authors.lastname: Siebert
authors.affiliation: 1] Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany [2] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Goldmann
authors.affiliation: 1] Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany [2] Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Ole
authors.initials: O
authors.lastname: Ammerpohl
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000368:Aged; D016477:Artifacts; D002021:Buffers; D018196:Carcinoma, Adenosquamous / Q000378:metabolism / Q000473:pathology; D018287:Carcinoma, Large Cell / Q000378:metabolism / Q000517:prevention & control; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D015925:Cryopreservation; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000737:chemistry; D006531:HEPES / Q000737:chemistry; D006801:Humans; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D046888:Tissue Array Analysis; D016707:Tissue Fixation; D014021:Tissue Preservation
chemical_list: D002021:Buffers; D004273:DNA, Neoplasm; D018698:Glutamic Acid; D006531:HEPES