Laboratory for Electron Microscopy, Bijenicka 54, Rudjer Boskovic Institute, HR-10000 Zagreb, Croatia.
J Immunol Methods. 2010 Jul 31;359(1-2):42-6. doi: 10.1016/j.jim.2010.05.010. Epub 2010 Jun 4.
Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.
阐明人类淋巴瘤发展过程中涉及的分子途径需要有效的方法来解决淋巴结中的基因表达问题。这些恶性肿瘤中转录因子的表达研究有助于理解在肿瘤转化和淋巴瘤发展过程中发生的变化。切除的淋巴结通常在福尔马林中固定并嵌入石蜡中用于诊断，并储存在许多医院的病理档案中。这些组织是研究的宝贵资源，因为它们允许回顾性研究涵盖广泛的人类淋巴瘤，甚至包括较不常见的类型。逆转录聚合酶链反应（RT-PCR）是一种常用的基因表达分析方法，从淋巴结中分离 RNA 的可重复协议是这些研究的必然要求。然而，福尔马林固定和石蜡包埋会干扰 RNA 的质量，特别是从淋巴结中分离时，淋巴结是最脆弱的淋巴组织。我们在这里介绍了一种从福尔马林固定石蜡包埋的淋巴结中分离 RNA 的简单快速方法，该方法可成功应用于 RT-PCR 以及定量 RT-PCR 分析。我们测试了多种分离试剂，并结合了一系列因素，以获得高质量的 RNA，用于人类淋巴瘤样本中基因表达的回顾性研究。我们从 FFPE 中提取 RNA 的改良方法基于 qPCR 数据提供了更高的产量和纯度。
