Tomlins Scott A, Mehra Rohit, Rhodes Daniel R, Shah Rajal B, Rubin Mark A, Bruening Eric, Makarov Vladimir, Chinnaiyan Arul M
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.
Neoplasia. 2006 Feb;8(2):153-62. doi: 10.1593/neo.05754.
Expression profiling of clinically obtainable tumor specimens has been hindered by the need for microgram quantities of RNA. In vitro transcription (IVT)-based amplifications are most commonly used to amplify small quantities of RNA for microarray analysis. However, significant drawbacks exist with IVT-based amplification, and the need for alternative amplification methods remains. Herein, we validate whole transcriptome amplification (WTA), an exponential amplification technique that produces cDNA libraries and amplified target in 3 to 4 hours from nanogram quantities of total RNA using a combination of cDNA microarrays and quantitative polymerase chain reaction (PCR). We demonstrate that WTA material can serve as a "molecular archive" because a WTA cDNA library can be faithfully amplified through multiple rounds of PCR amplification, allowing it to serve as a bankable and distributable resource. To demonstrate applicability, WTA was combined with laser capture microdissection to profile frozen prostate tissues. Unlike most IVT-based and exponential amplification techniques, WTA does not depend on the presence of a poly-A tail. Thus, we demonstrate that WTA is compatible with artificially degraded RNA and RNA isolated from formalin-fixed paraffin-embedded tissues. Taken together, WTA represents a versatile approach to profile and archive cDNA from minute tumor samples and is compatible with partially degraded RNA.
临床可获取肿瘤标本的表达谱分析一直受到所需微克级RNA量的阻碍。基于体外转录（IVT）的扩增是最常用于扩增少量RNA以进行微阵列分析的方法。然而，基于IVT的扩增存在显著缺点，因此对替代扩增方法的需求依然存在。在此，我们验证了全转录组扩增（WTA），这是一种指数扩增技术，它使用cDNA微阵列和定量聚合酶链反应（PCR）相结合的方法，在3至4小时内从纳克级的总RNA中产生cDNA文库并扩增靶标。我们证明WTA材料可作为一种“分子档案”，因为WTA cDNA文库可通过多轮PCR扩增得到忠实扩增，使其成为一种可储存和分发的资源。为证明其适用性，WTA与激光捕获显微切割技术相结合，对冷冻前列腺组织进行分析。与大多数基于IVT的指数扩增技术不同，WTA不依赖于poly-A尾的存在。因此，我们证明WTA与人工降解的RNA以及从福尔马林固定石蜡包埋组织中分离的RNA兼容。综上所述，WTA是一种从微小肿瘤样本中分析和存档cDNA的通用方法，并且与部分降解的RNA兼容。
