PDF(Pubmed)
Abstract：
Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.
摘要：
福尔马林固定石蜡包埋（FFPE）样品是许多毒理学和临床研究中唯一剩余的生物档案。然而，由于福尔马林固定的核酸损伤，它们在基因组学中的应用受到限制。具有高度降解的RNA的较老的FFPE样品提出了特别困难的技术挑战。基于探针的靶向测序技术显示出解决此问题的希望，但尚未与标准全基因组RNA测序（RNA-Seq）方法直接比较。在这项研究中,我们使用靶向重测序(TempO-Seq)和全基因组RNA-Seq方法评估了储存超过20年的配对冷冻(FROZ)和FFPE肝脏样本的剂量依赖性转录变化.最初从暴露于参考化学品（二氯乙酸，DCA），以0、198、313和427mg/kg-天（n=6/剂量）的浓度饮用水连续6天。TempO-Seq在匹配的FFPE和FROZ样品之间的差异表达基因（DEGs）中显示出高度重叠，并且在两个最高剂量水平的DCA与控制（R2≥0.94）。同样，TempO-SeqFFPE和RNA-SeqFROZ结果的倍数变化值高度一致(R2≥0.92).相比之下,与FROZRNA-Seq相比,RNA-SeqFFPE样品显示很少重叠的DEGs(对于所有剂量组≤5)。基因集DCA依赖性变化的建模确定了TempO-SeqFROZ和FFPE样品的基准剂量，范围为RNA-SeqFROZ样品的1.4倍（93.9mg/kg-d），而RNA-SeqFFPE样品高3.3倍（310.3mg/kg-d）。这项工作表明，靶向测序可以提供一种更可靠的方法来定量来自老化的归档FFPE样品的基因表达谱。
