Data from: Demodifying RNA for transcriptomic analyses of archival formalin-fixed paraffin-embedded samples

Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and transcriptional responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (±organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by >1.5-fold and >3.0-fold, respectively, versus 3F. Post-sequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77-84%) and enriched pathways (91-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.存档福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织样本为基因组学研究提供了体量庞大但尚未被充分开发的宝贵资源。限制FFPE样本应用的核心技术难题在于RNA的产出量与质量。本研究针对经福尔马林固定后发生高度断裂与交联的RNA，评估了多种脱修饰处理方法。本研究的主要评价终点包括RNA回收率、RNA测序质量指标，以及受试参考化学品苯巴比妥（phenobarbital, PB）诱导的转录应答。将来自对照组与苯巴比妥组的冷冻小鼠肝脏样本（每组n=6）均分后，分别按以下方式保存3个月：冷冻组（frozen, FR）、70%乙醇组（70% ethanol, OH）、10%缓冲福尔马林固定18小时后转入乙醇组（10% buffered formalin for 18 hours followed by ethanol, 18F），以及10%缓冲福尔马林直接固定组（10% buffered formalin, 3F）。将OH、18F与3F组的样本制备为FFPE组织块并切片，用于RNA提取。3F组的额外切片则接受以下脱修饰处理方案，以减轻RNA损伤：采用Tris-乙酸-EDTA缓冲液进行短时加热孵育；使用两种不同的RNA提取试剂盒，搭配有机催化剂进行过夜加热孵育；或不添加有机催化剂进行过夜加热孵育。采用Illumina HiSeq 2500测序平台，对核糖体RNA去除、链特异性总RNA文库进行构建与测序。与3F组相比，过夜孵育（添加/不添加有机催化剂）可使RNA产出量提升3倍以上，RNA完整性数值与片段分析值分别提升1.5倍以上与3.0倍以上。测序后质量指标同样显示，过夜孵育组的基因覆盖偏倚与缺失率均有所降低。所有脱修饰组与FR组的差异表达基因重叠率（77%~84%）以及富集通路重叠率（91%~97%）均有所提升，其中有机催化剂组的重叠率最高。本研究结果表明，仅需对RNA提取方法进行简单优化，即可有效提升FFPE样本的基因组学分析质量。