title: MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability.
abstract: BACKGROUND
  Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation.
  
  
  METHODS
  High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering.
  
  
  RESULTS
  We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results.
  
  
  CONCLUSIONS
  We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.
authors.affiliation: Department of Oncology, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730, Herlev, Denmark. Mogens.karsboel.boisen@regionh.dk.
authors.forename: Mogens Karsbøl
authors.initials: MK
authors.lastname: Boisen
authors.affiliation: Statistics, Bioinformatics and Registry, Danish Cancer Society Research Center, Copenhagen, Denmark. chrdehl@cancer.dk.
authors.forename: Christian
authors.initials: C
authors.lastname: Dehlendorff
authors.affiliation: Department of Pathology, Herlev and Gentofte Hospital, Herlev, Denmark. Dorte.Linnemann@regionh.dk.
authors.forename: Dorte
authors.initials: D
authors.lastname: Linnemann
authors.affiliation: Department of Surgical Gastroenterology, Rigshospitalet, Copenhagen, Denmark. Nicolai.Aagaard.Schultz.01@regionh.dk.
authors.forename: Nicolai Aagaard
authors.initials: NA
authors.lastname: Schultz
authors.affiliation: Department of Oncology, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730, Herlev, Denmark. benny.vittrup.jensen@regionh.dk.
authors.forename: Benny Vittrup
authors.initials: BV
authors.lastname: Jensen
authors.affiliation: Department of Pathology, Herlev and Gentofte Hospital, Herlev, Denmark. Estrid.Hoegdall@regionh.dk.
authors.forename: Estrid Vilma Solyom
authors.initials: EV
authors.lastname: Høgdall
authors.affiliation: Department of Oncology, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730, Herlev, Denmark. julia.johansen@post3.tele.dk.|Department of Medicine, Herlev and Gentofte Hospital, Herlev, Denmark. julia.johansen@post3.tele.dk.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. julia.johansen@post3.tele.dk.
authors.forename: Julia Sidenius
authors.initials: JS
authors.lastname: Johansen
mesh_terms: D000367:Age Factors; D014408:Biomarkers, Tumor / Q000235:genetics; D016000:Cluster Analysis; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling / Q000592:standards*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D035683:MicroRNAs