National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27709.
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877.
Toxicol Sci. 2019 Aug 1;170(2):357-373. doi: 10.1093/toxsci/kfz113.
Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100-300 nucleotides in size (DV100-300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.
福尔马林固定石蜡包埋 (FFPE) 组织为毒理基因组学研究提供了重要资源。然而，从存档的 FFPE 标本中获得的 RNA 的完整性或质量的变化可能导致不可靠的数据和浪费资源，并且用于测量 RNA 完整性的标准方案不能充分评估 FFPE RNA 的适用性。本研究的主要目标是确定改进的方法来评估用于全基因组测序的 FFPE RNA 质量。我们检查了根据块中的年龄和福尔马林中的时间在冷冻和 FFPE 样本对中进行的 RNA 质量指标，这些样本的质量各不相同。通过 RNA 完整性数 (RIN)、称为石蜡包埋 RNA 度量的改良 RIN、大小 >100-300 个核苷酸的 RNA 片段的百分比 (DV100-300) 和 2 种基于定量 PCR 的方法来测量 RNA 质量。将此信息与测序读质量、映射和基因检测相关联。在基于片段的方法中，与 RIN 或石蜡包埋 RNA 度量相比，DV 和基于 PCR 的指标在确定测序成功方面提供了更多信息。在低质量和高质量的 FFPE 样本中，至少 80% 的 >100 个核苷酸的 RNA 片段 (DV100 > 80) 提供了测序时基因多样性和读计数的最佳指示。基于 PCR 的方法进一步显示，与样本年龄和福尔马林时间相关的靶基因的可扩增 RNA 定量减少，为测序的 FFPE RNA 输入量提供了信息。这些结果应该有助于筛选和优先考虑存档的 FFPE 样本，以进行基因表达的回顾性分析。
