Nojima Hiroshi, Tougan Takahiro
Department of Molecular Genetics and DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Methods Mol Biol. 2011;729:15-35. doi: 10.1007/978-1-61779-065-2_2.
Unlike exponential amplification using polymerase chain reaction (PCR), linear RNA amplification using T7 RNA polymerase is advantageous for genome-wide analysis of gene expression and for cDNA library preparation from single-cell quantities of RNA. However, the use of RNA polymerase requires a large amount of RNA, as the optimum concentration of the substrate (mRNA), or the Michaelis constant (K(m)), is one millionfold higher than the single-cell amount of mRNA. To circumvent this K(m) problem, we designed a small mRNA-like dummy molecule, termed chum-RNA, which can be easily removed after the completion of the reaction. Chum-RNA allowed the preparation of a high-quality cDNA library from single-cell quantities of RNA after four rounds of T7-based linear amplification, without using PCR amplification. The use of chum-RNA may also facilitate quantitative reverse-transcription (qRT)-PCR from small quantities of substrate.
与使用聚合酶链反应（PCR）进行指数扩增不同，使用T7 RNA聚合酶进行线性RNA扩增有利于全基因组基因表达分析以及从单细胞量的RNA制备cDNA文库。然而，RNA聚合酶的使用需要大量RNA，因为底物（mRNA）的最佳浓度或米氏常数（K(m)）比单细胞量的mRNA高一百万倍。为了解决这个K(m)问题，我们设计了一种小的mRNA样虚拟分子，称为chum-RNA，它在反应完成后可以很容易地去除。经过四轮基于T7的线性扩增后，chum-RNA使得能够从单细胞量的RNA制备高质量的cDNA文库，而无需使用PCR扩增。chum-RNA的使用也可能有助于从小量底物进行定量逆转录（qRT）-PCR。
