Kita-Matsuo Hiroko, Yukinawa Naoto, Matoba Ryo, Saito Sakae, Oba Shigeyuki, Ishii Shin, Kato Kikuya
Taisho Laboratory of Functional Genomics, Nara Institute of Science Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Anal Biochem. 2005 Apr 1;339(1):15-28. doi: 10.1016/j.ab.2004.11.014.
Adaptor-tagged competitive polymerase chain reaction (ATAC-PCR) is an advanced version of quantitative competitive PCR characterized by the addition of unique adaptors to different cDNA samples. It is currently the only quantitative PCR technique that enables large-scale gene expression analysis. Multiplex application of ATAC-PCR employs seven adaptors, two or three of which are used as controls to generate a calibration curve. The characteristics of the ATAC-PCR method for large-scale data production, including any adaptor- and gene-dependent amplification biases, were evaluated by using this method to analyze the expression of 384 mouse brain genes. Short adaptors tended to amplify at higher efficiency than did long adaptors. The population of genes with a high amplification bias increased with the use of short adaptors. Subtracting the median value of all adaptor-dependent biases could reduce this bias; the majority of genes displayed a small gene-dependent bias, which facilitated reliable quantification. We modified ATAC-PCR to estimate molecular numbers of transcripts by introducing synthetic standards. This modification demonstrated that gene expression levels in mammalian cells are varied over seven orders of magnitude.
衔接子标记竞争聚合酶链反应（ATAC-PCR）是定量竞争PCR的一种改进形式，其特点是在不同的cDNA样本中添加独特的衔接子。它是目前唯一能够进行大规模基因表达分析的定量PCR技术。ATAC-PCR的多重应用使用七种衔接子，其中两到三种用作对照以生成校准曲线。通过使用该方法分析384个小鼠脑基因的表达，评估了ATAC-PCR方法用于大规模数据生成的特性，包括任何与衔接子和基因相关的扩增偏差。短衔接子的扩增效率往往高于长衔接子。随着短衔接子的使用，具有高扩增偏差的基因数量增加。减去所有与衔接子相关偏差的中值可以减少这种偏差；大多数基因显示出较小的与基因相关的偏差，这有助于进行可靠的定量。我们通过引入合成标准品对ATAC-PCR进行了改进，以估计转录本的分子数。这种改进表明，哺乳动物细胞中的基因表达水平在七个数量级上有所不同。
