Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs.
Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs.
复制标题
DOI：
10.1186/1471-2164-9-574
复制
发表时间：
2008-11-29
期刊：
影响因子：
4.4
通讯作者：
Ito, Takashi
中科院分区：
文献类型：
--
作者：
Miura, Fumihito;Kawaguchi, Noriko;Yoshida, Mikio;Uematsu, Chihiro;Kito, Keiji;Sakaki, Yoshiyuki;Ito, Takashi
描述转录组的理想格式是以每个细胞的单个mRNA的绝对数量为尺度测量其组成。这不仅有助于精确掌握转录组的结构，还有助于加速数据交换和整合。我们设想了基因组DNA和cDNA之间的竞争PCR。由于前者包含完全相同拷贝数的每个基因，它可以作为后者获得转录组化学计量组成数据的理想归一化标准。然后，该数据可以容易地转换为提供有适当校准的绝对定量数据。为了实现这一想法，我们改进了衔接子标记的竞争性PCR，最初开发用于每个cDNA的3 '端限制性片段的相对定量，使得它可以应用于任何限制性片段。我们证明了这种“广义”衔接子标记的竞争性PCR（GATC-PCR）可以在基因组DNA和cDNA之间进行，以准确地测量芽殖酵母酿酒酵母中每个mRNA的绝对表达水平。此外，我们构建了一个大规模的GATC-PCR系统来测量5，038个基因的绝对表达水平，以表明酵母每个细胞含有超过30，000个mRNA分子拷贝。我们开发了一种GATC-PCR方法，通过竞争性扩增每个基因的基因组和cDNA拷贝来准确测量mRNA的绝对表达水平。GATC-PCR对芽殖酵母转录组的大规模应用表明，它是以前估计的两倍或更多。该方法灵活适用于mRNA绝对表达水平的靶向和全基因组分析。
An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration. We conceived an idea of competitive PCR between genomic DNA and cDNA. Since the former contains every gene exactly at the same copy number, it can serve as an ideal normalization standard for the latter to obtain stoichiometric composition data of the transcriptome. This data can then be easily converted to absolute quantification data provided with an appropriate calibration. To implement this idea, we improved adaptor-tagged competitive PCR, originally developed for relative quantification of the 3'-end restriction fragment of each cDNA, such that it can be applied to any restriction fragment. We demonstrated that this "generalized" adaptor-tagged competitive PCR (GATC-PCR) can be performed between genomic DNA and cDNA to accurately measure absolute expression level of each mRNA in the budding yeast Saccharomyces cerevisiae. Furthermore, we constructed a large-scale GATC-PCR system to measure absolute expression levels of 5,038 genes to show that the yeast contains more than 30,000 copies of mRNA molecules per cell. We developed a GATC-PCR method to accurately measure absolute expression levels of mRNAs by means of competitive amplification of genomic and cDNA copies of each gene. A large-scale application of GATC-PCR to the budding yeast transcriptome revealed that it is twice or more as large as previously estimated. This method is flexibly applicable to both targeted and genome-wide analyses of absolute expression levels of mRNAs.
登录
查看更多内容
DOI：
10.1073/pnas.93.11.5208
发表时间：
1996-05-28
影响因子：
11.1
作者：
Iyer, V;Struhl, K
通讯作者：
Struhl, K
影响因子：
7.7
作者：
van de Peppel, J;Kemmeren, P;Holstege, FCP
通讯作者：
Holstege, FCP
影响因子：
3.2
作者：
Wei, Y;Lee, JM;LaRossa, RA
通讯作者：
LaRossa, RA
Priperstation：人类基因组的高度特定的多重基因组PCR底漆设计服务器。
影响因子：
14.9
作者：
Yamada T;Soma H;Morishita S
通讯作者：
Morishita S
DOI：
10.1073/pnas.0605645103
发表时间：
2006-11-21
影响因子：
11.1
作者：
Miura, Fumihito;Kawaguchi, Noriko;Ito, Takashi
通讯作者：
Ito, Takashi