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

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.
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Name(s): Vukmirovic, Milica, author
Herazo-Maya, Jose D, author
Blackmon, John, author
Skodric-Trifunovic, Vesna, author
Jovanovic, Dragana, author
Pavlovic, Sonja, author
Stojsic, Jelena, author
Zeljkovic, Vesna, author
Yan, Xiting, author
Homer, Robert, author
Stefanovic, Branko, author
Kaminski, Naftali, author
Type of Resource: text
Genre: Journal Article
Text
Date Issued: 2017-01-12
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: 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.
Identifier: FSU_pmch_28081703 (IID), 10.1186/s12890-016-0356-4 (DOI), PMC5228096 (PMCID), 28081703 (RID), 28081703 (EID), 10.1186/s12890-016-0356-4 (PII)
Keywords: DEGs, FFPE, Idiopathic Pulmonary Fibrosis, MMP7, Microarray, NanoString nCounter®, Network, Pathways, RNA-Seq, Validation
Grant Number: KL2 TR001862, R01 HL127349, UL1 TR001863
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228096.
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_pmch_28081703
Owner Institution: FSU
Is Part Of: BMC pulmonary medicine.
1471-2466
Issue: iss. 1, vol. 17

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Vukmirovic, M., Herazo-Maya, J. D., Blackmon, J., Skodric-Trifunovic, V., Jovanovic, D., Pavlovic, S., … Kaminski, N. (2017). 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. Retrieved from http://purl.flvc.org/fsu/fd/FSU_pmch_28081703