An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
复制标题
一种改进的单细胞 cDNA 扩增方法，用于高效的高密度寡核苷酸微阵列分析。
DOI：
10.1093/nar/gkl050
复制
发表时间：
2006
影响因子：
14.9
通讯作者：
Saitou M
中科院分区：
文献类型：
--
作者：
Kurimoto K;Yabuta Y;Ohinata Y;Ono Y;Uno KD;Yamada RG;Ueda HR;Saitou M
对发育或成年期的小但必需的细胞群体（例如体干细胞）的系统水平的理解需要对全基因组基因表达进行准确的定量监测，理想情况下是从单细胞进行。我们在这里报告了一种策略，从单细胞中全局扩增mRNA的高定量高密度寡核苷酸微阵列分析，结合了少量的定向PCR循环与随后的线性扩增。使用这种策略，基因表达谱的表示和单个实验之间的再现性从原始方法明确地改进，沿着具有高覆盖率和准确性。将该方法直接应用于胚胎第3.5天（E）小鼠胚泡未分化内细胞团中的单细胞，发现存在两个细胞群，一个具有原始内胚层（PE）表达，另一个具有多能外胚层样基因表达。这两个群体之间差异表达的基因在一天后（E4.5）的胚胎中形态分化的PE和上胚层中保存良好，表明该方法成功地检测到看似同质的细胞群体之间在单细胞水平上基因表达的细微但本质的差异。这项研究提供了一种分析医学以及神经，干细胞和发育生物学中生物物理事件的策略，其中少量独特或患病细胞起着关键作用。
A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.
登录
查看更多内容
影响因子：
14.9
作者：
Baugh, L. R.;Hill, A. A.;Hunter, Craig P.
通讯作者：
Hunter, Craig P.
影响因子：
56.9
作者：
Golub, TR;Slonim, DK;Lander, ES
通讯作者：
Lander, ES
影响因子：
5.3
作者：
ARCECI, RJ;KING, AAJ;WILSON, DB
通讯作者：
WILSON, DB
影响因子：
1.5
作者：
Goldin, SN;Papaioannou, VE
通讯作者：
Papaioannou, VE
影响因子：
6.9
作者：
Brandt, S;Kloska, S;Kehr, J
通讯作者：
Kehr, J