Ma Zhihai, Lui Weng-Onn, Fire Andrew, Dadras Soheil S
Departments of Pathology, Stanford University School of Medicine,Stanford, California, USA.
J Mol Diagn. 2009 Sep;11(5):420-9. doi: 10.2353/jmoldx.2009.090041.
Archived formalin-fixed, paraffin-embedded human tumors are widely available and represent a unique source of morphologically defined material. Formalin-fixed, paraffin-embedded tissue is known to contain a wealth of molecular information in the form of microRNAs (miRNAs), which could be correlated with clinical outcome for improved prognostication and/or treatment response. miRNAs are endogenous, noncoding RNAs ( approximately 22 nucleotides) and may function as tumor suppressors or oncogenes. A reliable, robust methodology is needed to take full advantage of archived human cancers, especially for those where fresh-frozen tumor banks are unavailable, for example, malignant melanoma. To this end, we applied a simple-to-use protocol for extracting total RNA from various formalin-fixed, paraffin-embedded specimens (colon, liver, prostate, thyroid, uterus, and skin), optimized for small RNA recovery. Using a "poison primer" strategy (ie, primer silencing), we blocked the amplification of ribosomal RNA, enabling the successful sequencing of 17 novel and 53 known miRNAs (including small RNAs) from 10-year-old archived normal skin, cutaneous scalp melanoma, and sentinel lymph nodes (both negative and positive for metastasis) excised from a 52-year-old man. The cloning incidence provided an estimation of the level of specific miRNA expression, which was confirmed by Northern analysis and quantitative real-time polymerase chain reaction. This methodology can therefore be used to facilitate miRNA discovery from archived human cancers.
存档的福尔马林固定、石蜡包埋的人类肿瘤样本广泛可得，是形态学明确材料的独特来源。福尔马林固定、石蜡包埋的组织已知含有以微小RNA（miRNA）形式存在的丰富分子信息，这些信息可与临床结果相关联，以改善预后和/或治疗反应。miRNA是内源性非编码RNA（约22个核苷酸），可作为肿瘤抑制因子或癌基因发挥作用。需要一种可靠、稳健的方法来充分利用存档的人类癌症样本，特别是对于那些没有新鲜冷冻肿瘤库的癌症，例如恶性黑色素瘤。为此，我们应用了一种简单易用的方案，从各种福尔马林固定、石蜡包埋的标本（结肠、肝脏、前列腺、甲状腺、子宫和皮肤）中提取总RNA，并针对小RNA回收进行了优化。使用“毒引物”策略（即引物沉默），我们阻断了核糖体RNA的扩增，从而成功地从一名52岁男性的10年前存档的正常皮肤、头皮皮肤黑色素瘤以及前哨淋巴结（转移阴性和阳性）中测序出17种新的和53种已知的miRNA（包括小RNA）。克隆发生率提供了特定miRNA表达水平的估计，这通过Northern分析和定量实时聚合酶链反应得到了证实。因此，这种方法可用于促进从存档的人类癌症中发现miRNA。
