Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1–240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6–72 h) for cancer patients.

中文翻译：

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福尔马林固定时间对福尔马林固定石蜡包埋肝癌组织RNA质量和数量的影响

分析福尔马林固定石蜡包埋 (FFPE) 组织中的 RNA 样本对于精准医疗至关重要。我们研究了在福尔马林中固定不同时间的 FFPE 肿瘤组织的 RNA 数量和质量，并比较了下一代测序 (NGS) 的测序指标。将肝细胞癌 (HCC) 组织固定在 10% 中性缓冲福尔马林中（1-240 小时），并制备 FFPE 块。提取总RNA，并使用NanoDrop、Qubit 和生物分析仪评估其数量和质量。制备测序文库后，在 Oncomine Dx Multi-CDx 系统上进行 NGS。所有样品的总 RNA 产量均满足 NGS 所需的阈值，但较长的固定时间导致总 RNA 和长 RNA 片段 (>200 nt) 产量下降。NGS 分析显示，随着固定时间的延长，RNA 中内部对照基因的测序读数会减少。与从立即制备的 FFPE 块中获得的 RNA 相比，从储存 500 天的 FFPE 块中提取的 RNA 的 RNA 产量和质量有所降低。总之，应避免短固定和过度固定，因为它们会对测序质量产生负面影响。固定过程应在癌症患者推荐的指南（6-72 小时）内迅速完成。