Quantitative analysis of mRNA amplification by in vitro transcription
Quantitative analysis of mRNA amplification by in vitro transcription
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DOI：
10.1093/nar/29.5.e29
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发表时间：
2001-03-01
影响因子：
14.9
通讯作者：
Hunter, Craig P.
中科院分区：
文献类型：
--
作者：
Baugh, L. R.;Hill, A. A.;Hunter, Craig P.
动物系统中有效的转录物分析需要分离同质组织或细胞，然后进行忠实的 mRNA 扩增。据报道，基于 cDNA 合成和体外转录的线性扩增可以维持 mRNA 水平的代表性，然而，缺乏证明这一点的定量数据以及对固有局限性的描述。我们表明，已发布的方案除了放大真实目标 mRNA 之外，还产生了独立于模板的产物，从而降低了最终产物的比活性。我们描述了一种改进的扩增方案，该方案最大限度地减少了模板无关产物的生成，因此可以从 100 ng 总 RNA 中生成所需微克量的信息衍生材料。应用第二轮嵌套的 cDNA 合成和体外转录将所需的起始材料减少到 2 ng 总 RNA。对这些产品对秀丽隐杆线虫 Affymetrix GeneChips 的定量分析表明，这种扩增不会降低总体灵敏度，并且对保真度仅产生很小的影响。
Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.