title: Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.
abstract: Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Hedegaard
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Thorsen
authors.affiliation: AROS Applied Biotechnology A/S, Science Park Skejby, Aarhus, Denmark.
authors.forename: Mette Katrine
authors.initials: MK
authors.lastname: Lund
authors.affiliation: CLC bio, Aarhus, Denmark.
authors.forename: Anne-Mette K
authors.initials: AM
authors.lastname: Hein
authors.affiliation: Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Stephen Jacques
authors.initials: SJ
authors.lastname: Hamilton-Dutoit
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Vang
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Iver
authors.initials: I
authors.lastname: Nordentoft
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Karin
authors.initials: K
authors.lastname: Birkenkamp-Demtröder
authors.affiliation: AROS Applied Biotechnology A/S, Science Park Skejby, Aarhus, Denmark.
authors.forename: Mogens
authors.initials: M
authors.lastname: Kruhøffer
authors.affiliation: Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Hager
authors.affiliation: CLC bio, Aarhus, Denmark.
authors.forename: Bjarne
authors.initials: B
authors.lastname: Knudsen
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Claus Lindbjerg
authors.initials: CL
authors.lastname: Andersen
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Karina Dalsgaard
authors.initials: KD
authors.lastname: Sørensen
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Jakob Skou
authors.initials: JS
authors.lastname: Pedersen
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Torben Falck
authors.initials: TF
authors.lastname: Ørntoft
authors.affiliation: Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Dyrskjøt
mesh_terms: D015925:Cryopreservation; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D059472:Exome / Q000235:genetics; D005557:Formaldehyde / Q000494:pharmacology; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D012313:RNA; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins