Baugh L R, Hill A A, Brown E L, Hunter C P
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Nucleic Acids Res. 2001 Mar 1;29(5):E29. doi: 10.1093/nar/29.5.e29.
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.
在动物系统中进行有效的转录本分析，需要先分离出均质的组织或细胞，然后进行可靠的mRNA扩增。据报道，基于cDNA合成和体外转录的线性扩增能够维持mRNA水平的代表性，然而，缺乏证明这一点的定量数据以及对其固有局限性的描述。我们发现，已发表的方案除了扩增真正的目标mRNA外，还会产生与模板无关的产物，从而降低了最终产物的比活性。我们描述了一种改良的扩增方案，该方案可将与模板无关的产物生成量降至最低，因此能够从100 ng总RNA中生成所需的微克量的源自mRNA的材料。第二轮嵌套式cDNA合成和体外转录的应用将所需的起始材料减少至2 ng总RNA。对秀丽隐杆线虫Affymetrix基因芯片上的这些产物进行定量分析表明，这种扩增不会降低总体灵敏度，对保真度的影响也很小。
