The results obtained from FFPE tissue are highly comparable to FF tissues. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. 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. 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). 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. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Only 92 differentially expressed genes changed in opposite directions. The overlap of differentially expressed genes was very high (760 increased and 1,413 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). 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. TopHat2 was used to map sequencing reads to the human genome. 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. 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.

A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology.

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