RNA amplification for successful gene profiling analysis.
RNA amplification for successful gene profiling analysis.
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DOI：
10.1186/1479-5876-3-28
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发表时间：
2005-07-25
影响因子：
7.4
通讯作者：
Wang E
中科院分区：
文献类型：
--
作者：
Wang E
临床样本的研究往往受到可供研究的材料数量的限制。虽然蛋白质不能以其自然形式繁殖，但DNA和RNA可以从小样本中扩增出来，用于高通量分析。因此，在几乎没有可用的材料的情况下，基因研究提供了筛选人类病理学新见解的最佳机会。精确估计给定样本中的DNA拷贝数是必要的。然而，大多数研究调查的是静态变量，如患者的遗传背景或病理标本中的突变，而不需要评估整个基因组中不同基因之间的表达比例。DNA样本的比较基因组杂交是这一规则的一个粗略例外，因为基因组的扩增或缺失是在不同的样本之间直接进行比较的。然而，对于基因表达分析，准确估计不同RNA转录本的比例表达是至关重要的，因为这些比例直接通过调节蛋白质表达来控制细胞功能。此外，对不同时间点的相对RNA表达的比较估计描绘了细胞对环境刺激的反应，间接地告知在生理或病理条件下影响特定组织的更广泛的生物事件。细胞的这种认知反应类似于脑电模式的检测，脑电模式可以告知大脑对外部刺激的反应状态。随着我们在全球水平上了解人类病理生理学的需求增加，高保真信使RNA扩增技术的开发和改进在过去十年中已经成为人们日益感兴趣的焦点。增加RNA丰度的需求不仅满足了基因特异性扩增的需要，而且最重要的是满足了全球转录组广泛、无偏见的扩增的需要。现在，基因特异性的、无偏见的转录组广泛扩增准确地保持了给定样本内所有RNA物种之间的比例。这使得可以利用通过细针抽吸(FNA)或细胞学洗涤等微创方法获得的临床材料进行高通量功能基因组研究。这篇综述对这一主题的文献进行了全面和最新的讨论，并批判性地讨论了主要方法、陷阱，并为在临床样本中成功地无偏扩增整个转录组提供了实用建议。
The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene-specific, unbiased transcriptome wide amplification accurately maintains proportionality among all RNA species within a given specimen. This allows the utilization of clinical material obtained with minimally invasive methods such as fine needle aspirates (FNA) or cytological washings for high throughput functional genomics studies. This review provides a comprehensive and updated discussion of the literature in the subject and critically discusses the main approaches, the pitfalls and provides practical suggestions for successful unbiased amplification of the whole transcriptome in clinical samples.
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