Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.
Background: 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...
| Publicado en: | BMC Pulmonary Medicine Vol. 17; pp. 1 - 13 |
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| Autores principales: | , , , , , , , , , , , , |
| Formato: | research Journal Article |
| Publicado: |
BioMed Central
1/12/2017
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=120696880&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 120696880 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 14712466 1CIL jtl: BMC Pulmonary Medicine issn: 14712466 maglogo: N pubinfo: dt: 1/12/2017 vid: 17 pid: 24147 pub: BioMed Central artinfo: ui: 120696880 120696880 NLM28081703 120696880 10.1186/s12890-016-0356-4 NLM28081703 120696880 ppf: 1 ppct: 12 formats: tig: atl: Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis. aug: au: Vukmirovic, Milica Herazo-Maya, Jose D. Blackmon, John Skodric-Trifunovic, Vesna Jovanovic, Dragana Pavlovic, Sonja Stojsic, Jelena Zeljkovic, Vesna Xiting Yan Homer, Robert Stefanovic, Branko Kaminski, Naftali Yan, Xiting affil: Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA sug: subj: Lung Pathology RNA Analysis Idiopathic Pulmonary Fibrosis Gene Expression Profiling Idiopathic Pulmonary Fibrosis Pathology Paraffin Embedding Molecular Structure Freezing United States Human Peptide Hydrolases Child Signal Transduction Case Control Studies Sequence Analysis Microarray Analysis Validation Studies Comparative Studies Evaluation Research Multicenter Studies Child: 6-12 years ab: Background: 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.Results: 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.Conclusions: 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. pubtype: Academic Journal doctype: research Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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