Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray
Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray
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DOI：
10.1002/jnr.20533
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发表时间：
2005-06-15
影响因子：
4.2
通讯作者：
Tashiro, T
中科院分区：
文献类型：
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作者：
Takahashi, M;Kondoh, Y;Tashiro, T
DNA微阵列在神经科学中的使用一直局限于粗略筛选目的，主要是因为现有阵列的可靠性和灵敏度不够高。由于在大多数实验中通常只发现几百到一千个基因改变表达水平，我们试图通过以下策略来开发更定量的阵列：1)将基因限制在与实验系统相关的基因；2)设计特定分子特性的寡核苷酸探针，以便采用更严格的杂交条件；3)在一张玻片上放置6个探针并使用多个归一化基因；4)使用一种新型的镀金玻片，具有更高的S/N比。通过比较出生后第15天甲状腺功能减退小鼠和对照小鼠的小脑，从文献和实验中选择了与突触的构建和功能有关的基因(P15)。虽然所覆盖的基因数量还不多(172个基因)，但这样构建的定制阵列“SynaptoArrow”能够检测到+/-20%的基因表达比率差异。用突触阵列分析小鼠小脑的出生后发育，发现了一个总体的表达模式，在P7达到高峰，然后在P15下降，在P21之后部分恢复。P10清楚地标志着这个系统的初始生长阶段的结束和一个主要的转录转折点。这一结果表明，这样的定制数组在监测对正常发育程序的扰动方面应该是有用的。(C)2005年Wiley-Liss，Inc.
Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/- 20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this sytem. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program. (c) 2005 Wiley-Liss, Inc.