NCI CCR Sequencing Facility, Frederick National Laboratory for Cancer Research, Frederick, MD, United States of America.
Advanced Biomedical and Computational Sciences, Frederick National Laboratory for Cancer Research, Frederick, MD, United States of America.
PLoS One. 2019 May 6;14(5):e0216050. doi: 10.1371/journal.pone.0216050. eCollection 2019.
Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues.
福尔马林固定石蜡包埋（FFPE）组织是最广泛可用的临床标本之一。如果能将其作为转录组研究的 RNA 来源，将极大地促进基于人群的癌症研究。虽然初步研究表明 FFPE 组织可用于 RNA 测序，但这些标本的储存时间的影响仍需确定。我们进行了这项研究，以确定来自监测、流行病学和最终结果（SEER）登记处的存档 FFPE 高级卵巢浆液性腺癌组织中的 RNA 是否存在足够的数量和质量用于 RNA-Seq 分析。FFPE 组织从三个 SEER 地点获得，储存时间从 7 年到 32 年不等。提取、定量、质量评估，并进行 RNA-Seq（一种全转录组测序技术）。较长时间储存的 FFPE 标本的 RNA 样本质量较差，因为标本储存时间与片段分布值（DV）之间存在负相关。此外，RNA 样本污染是一个常见问题，67 个样本中有 41 个样本有 5%至 48%的细菌污染。然而，无论标本储存时间和细菌污染情况如何，60%的样本都能获得可用于基因表达定量的数据，鉴定出超过 10000 个基因，大多数生物学重复的相关性高于 0.7。这项研究表明，在储存库中储存长达 32 年且储存条件不同的 FFPE 高级卵巢浆液性腺癌标本是 RNA-Seq 的一种很有前途的 RNA 来源。我们还描述了在使用存档 FFPE 组织设计 RNA-Seq 研究时需要考虑的某些注意事项。
