Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis

BMC Pulmonary Medicine

Research Article

Jelena Stojsic¹  Branko Stefanovic²  John Blackmon²  Robert Homer³  Dragana Jovanovic⁴  Vesna Skodric-Trifunovic⁴  Vesna Zeljkovic⁵  Sonja Pavlovic⁶  Jose D. Herazo-Maya⁷  Xiting Yan⁷  Milica Vukmirovic⁷  Naftali Kaminski⁷ 

[1] Departement of Thoracopulmonary Pathology, Service of Pathology, Clinical Centre of Serbia, Belgrade, Serbia;Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA;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;Faculty of Medicine, University of Belgrade, Belgrade, Serbia;Clinic for Pulmonology, Clinical Center of Serbia, Belgrade, Serbia;Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia;Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia;Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT, USA;

关键词: Idiopathic Pulmonary Fibrosis;    FFPE;    RNA-Seq;    Microarray;    DEGs;    Validation;    Pathways;    Network;    MMP7;    NanoString nCounter®;   

DOI  :  10.1186/s12890-016-0356-4

 received in 2016-04-14, accepted in 2016-12-20,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundIdiopathic 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.ResultsWe 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.ConclusionsOur 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.

【 授权许可】

CC BY   
© The Author(s). 2017