Tougan Takahiro, Okuzaki Daisuke, Nojima Hiroshi
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nucleic Acids Res. 2008 Sep;36(15):e92. doi: 10.1093/nar/gkn420. Epub 2008 Jul 4.
Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 10(5) cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.
使用T7 RNA聚合酶进行线性RNA扩增在利用DNA微阵列进行全基因组基因表达分析中很有用，但从单细胞量的RNA制备cDNA文库仍需要使用聚合酶链反应（PCR）进行指数扩增。我们设计了一种名为chum-RNA的小RNA分子，它使我们能够在不使用PCR扩增的情况下，经过四轮基于T7的线性扩增后制备单细胞cDNA文库。Chum-RNA仅以0.49飞克的mRNA（730个mRNA分子）作为底物驱动cDNA合成，这个量相当于单个哺乳动物细胞中一小部分mRNA分子。对该文库的独立cDNA克隆（6.6×10⁵ cfu）的分析表明，在每一轮扩增过程中发生了30倍的RNA扩增。所得单细胞cDNA文库中mRNA的大小分布和代表性与百万细胞cDNA文库保持相似。Chum-RNA的使用也可能促进涉及其他DNA/RNA修饰酶的反应，这些酶的米氏常数（Kₘ）值约为1 mM，从而使它们在仅存在少量底物的情况下就能被激活。
