title: Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.
abstract: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.
  
  
  
  We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.
  
  
  
  Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.
authors.affiliation: Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA. milica.vukmirovic@yale.edu.
authors.forename: Milica
authors.identifier: 0000-0002-2025-3887
authors.initials: M
authors.lastname: Vukmirovic
authors.affiliation: Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Jose D
authors.initials: JD
authors.lastname: Herazo-Maya
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
authors.forename: John
authors.initials: J
authors.lastname: Blackmon
authors.affiliation: Faculty of Medicine, University of Belgrade, Belgrade, Serbia.|Clinic for Pulmonology, Clinical Center of Serbia, Belgrade, Serbia.
authors.forename: Vesna
authors.initials: V
authors.lastname: Skodric-Trifunovic
authors.affiliation: Faculty of Medicine, University of Belgrade, Belgrade, Serbia.|Clinic for Pulmonology, Clinical Center of Serbia, Belgrade, Serbia.
authors.forename: Dragana
authors.initials: D
authors.lastname: Jovanovic
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
authors.forename: Sonja
authors.initials: S
authors.lastname: Pavlovic
authors.affiliation: Departement of Thoracopulmonary Pathology, Service of Pathology, Clinical Centre of Serbia, Belgrade, Serbia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Stojsic
authors.affiliation: Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia.
authors.forename: Vesna
authors.initials: V
authors.lastname: Zeljkovic
authors.affiliation: Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Xiting
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.|Pathology and Laboratory Medicine Service, VA CT Healthcare System, West Haven, CT, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Homer
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
authors.forename: Branko
authors.initials: B
authors.lastname: Stefanovic
authors.affiliation: Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Naftali
authors.initials: N
authors.lastname: Kaminski
mesh_terms: D016022:Case-Control Studies; D002648:Child; D005615:Freezing; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000235:genetics* / Q000473:pathology*; D008168:Lung / Q000473:pathology*; D020783:Matrix Metalloproteinase 7 / Q000235:genetics; D046228:Microarray Analysis; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis*; D017423:Sequence Analysis, RNA; D014481:United States; D060449:Wnt Signaling Pathway
chemical_list: D012313:RNA; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7
keywords: DEGs; FFPE; Idiopathic Pulmonary Fibrosis; MMP7; Microarray; NanoString nCounter®; Network; Pathways; RNA-Seq; Validation