Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

中文翻译：

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优化的协议，可从适合于下一代测序的激光捕获显微切割的FFPE组织的小部分中分离RNA。

福尔马林固定石蜡包埋（FFPE）组织构成了用于生物医学研究的大量样品库。然而，到目前为止，由于化学RNA －蛋白质交联和RNA片段化，从FFPE组织中提取RNA已被证明具有挑战性，这两者都严重影响下游分析的RNA数量和质量。样本量非常小，例如，当执行激光捕获显微切割（LCM）分离细胞的特定亚群时，回收足够的RNA以便通过逆转录定量PCR（RT-qPCR）或下一代测序（NGS）进行分析变得非常重要麻烦和困难。我们使用LCM从FFPE犬乳腺肿瘤的临床标本中切除了匹配的癌症相关基质（CAS）和正常基质，并比较了常用的基于蛋白酶的RNA分离程序和经过改进的新技术，该技术另外还包含了聚焦超声处理步骤。我们成功地调整了使用聚焦超声的方案，以从少量去石蜡，染色的临床LCM样品中分离RNA。使用这种方法，我们发现与常规的基于蛋白酶的提取技术相比，总RNA产量可以提高8到12倍。出乎意料的是，使用这种新方法提取的RNA在质量上至少与旧方法相等，甚至没有优势，因为使用新方法后，RT-qPCR中的Cq值平均降低2.3倍。最后，我们证明了使用新方法提取的RNA在NGS中的表现也相当。我们提出了一种成功的分离方案，用于从困难且有限的FFPE组织样品中提取RNA，从而能够成功分析临床相关标本的一小部分。在档案FFPE组织的特定小区域中研究基因表达特征的可能性（通常需要大量高度相关的临床随访数据）为迄今为止难以分析的样品开辟了一个新的领域，如今这些样品可以进行研究。