Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.
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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DOI：
10.1186/s12890-016-0356-4
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发表时间：
2017-01-12
影响因子：
3.1
通讯作者：
Kaminski N
中科院分区：
文献类型：
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作者：
Vukmirovic M;Herazo-Maya JD;Blackmon J;Skodric-Trifunovic V;Jovanovic D;Pavlovic S;Stojsic J;Zeljkovic V;Yan X;Homer R;Stefanovic B;Kaminski N
特发性肺纤维化（IPF）是一种病因不明的致死性肺部疾病。IPF肺组织转录组学分析的主要局限性是依赖于速冻新鲜组织（FF）。在本项目中，我们试图确定使用RNA测序（RNA-Seq）的基因组规模转录物谱分析是否可应用于存档的福尔马林固定石蜡包埋（FFPE）IPF组织。我们从7个IPF和5个对照FFPE肺组织中分离总RNA，并在Illumina 2000 HiSeq上进行50个碱基对配对末端测序。TopHat 2用于将测序读数映射到人类基因组。平均每个样品映射约6200万个读段（约1.16亿个读段的53.4%）。IPF和对照组之间有4，131个基因差异表达（1，920个增加，2，211个减少（FDR < 0.05））。我们比较了我们的结果差异表达的基因计算从以前发表的数据集产生的FF组织上分析安捷伦微阵列（GSE 47460）。差异表达基因的重叠非常高（760个增加，1，413个减少，FDR < 0.05）。只有92个差异表达基因的变化方向相反。使用MetaCore进行的途径富集分析证实了许多IPF相关基因和途径，包括细胞外重塑、TGF-β和WNT。MMP 7是两个数据集中高度差异表达的基因，其基因网络分析揭示了RNA-Seq和微阵列数据中相同的典型途径和基因网络候选者。为了通过NanoString nCounter®进行验证，我们选择了在至少一个数据集中具有2倍变化的35个基因（10个不一致，10个仅在一个数据集中显著差异表达，以及15个一致基因）。对于每种类型的样品（FF对FFPE），用微阵列（r = 0.92）和RNA-Seq（r = 0.90）观察到倍数变化和FDR的高度一致性，并且不一致基因的数量减少到4个。我们的结果表明，从存档的FFPE肺组织中获得的RNA的RNA测序是可行的。从FFPE组织获得的结果与FF组织具有高度可比性。在存档的FFPE IPF组织上进行RNA测序的能力将大大提高组织活检用于IPF研究的可用性。本文的在线版本（doi：10.1186/s12890-016-0356-4）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/s12890-016-0356-4) contains supplementary material, which is available to authorized users.