Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6–7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

中文翻译：

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从激光捕获显微解剖的人和啮齿动物胰腺中回收高质量的 RNA

激光捕获显微切割 (LCM) 是一种强大的方法，可以分离特定细胞群以进行后续分析，例如基因表达谱分析，例如微阵列或核糖核酸 (RNA)-Seq。该技术已应用于冷冻和福尔马林固定石蜡包埋 (FFPE) 标本，在提取 RNA 的质量和数量方面具有不同的结果。该研究的目标是开发从 LCM 分离的朗格汉斯岛和胰管腺 (PDG) 中分离高质量 RNA 的方法。我们报告了冷冻切片的优化方案，通过在实验过程中的多个步骤添加 RNase 抑制剂，使用定量实时聚合酶链反应 (RT-PCR) 最大限度地减少 RNA 降解并最大限度地从样品中回收预期的转录本。该技术可重复提供完整的 RNA（RIN 值 6-7）。使用定量 RT-PCR，检测了 PDG 和胰岛中胰岛素、胰高血糖素、粘蛋白 6 (Muc6) 和细胞角蛋白 19 (CK-19) mRNA 的预期特征。所描述的冷冻胰腺组织实验方案也可能对其他具有中等至高水平内在核糖核酸酶 (RNase) 活性的组织有用。