Hedegaard Jakob, Thorsen Kasper, Lund Mette Katrine, Hein Anne-Mette K, Hamilton-Dutoit Stephen Jacques, Vang Søren, Nordentoft Iver, Birkenkamp-Demtröder Karin, Kruhøffer Mogens, Hager Henrik, Knudsen Bjarne, Andersen Claus Lindbjerg, Sørensen Karina Dalsgaard, Pedersen Jakob Skou, Ørntoft Torben Falck, Dyrskjøt Lars
Department of Molecular Medicine (MOMA), Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, Aarhus, Denmark.
AROS Applied Biotechnology A/S, Science Park Skejby, Aarhus, Denmark.
PLoS One. 2014 May 30;9(5):e98187. doi: 10.1371/journal.pone.0098187. eCollection 2014.
Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.
福尔马林固定、石蜡包埋（FFPE）组织是临床研究的宝贵资源。然而，从FFPE组织中提取的核酸会发生片段化和化学修饰，这使得它们在分子研究中的应用具有挑战性。我们分析了23个新鲜冷冻（FF）样本、35个FFPE样本和38对FF/FFPE配对样本，这些样本代表六种不同的人体组织类型（膀胱癌、前列腺癌和结肠癌；肝脏和结肠正常组织；反应性扁桃体），以研究FFPE样本在基于下一代测序（NGS）的回顾性和前瞻性临床研究中的潜在用途。我们比较了两种从FFPE组织中提取DNA的方法和三种提取RNA的方法，发现它们会影响核酸的数量和质量。从选定的FFPE样本和配对的FF/FFPE样本中提取的DNA和RNA用于外显子组和转录组分析。DNA外显子组测序文库的制备比RNA测序文库更具挑战性（成功率为29.5%），这可能是由于FFPE组织来源的DNA发生了修饰。从保存了二十年的FFPE组织中分离出的RNA仍然可以制备文库。使用CLC Bio基因组工作台对数据进行分析，结果显示FF和FFPE组织来源的核酸文库之间存在系统性差异。尽管如此，对DNA外显子组测序数据的成对分析表明，保存时间少于三年的FF和FFPE样本中，70-80%的变异具有一致性。RNA测序数据显示，FF/FFPE配对样本中的表达谱具有高度相关性（Pearson相关系数为0.90±0.05），与保存时间（长达244个月）和组织类型无关。我们确定了一组共1494个基因，无论组织类型如何，其在配对的FF和FFPE样本中的表达谱均存在显著差异。我们的结果很有前景，表明在无法获得FF样本的前瞻性和基于存档的回顾性研究中，NGS可用于研究FFPE样本。
