Using array hybridization to monitor gene expression at the single cell level
Using array hybridization to monitor gene expression at the single cell level
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DOI：
10.1093/jxb/erf093
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发表时间：
2002-12-01
影响因子：
6.9
通讯作者：
Kehr, J
中科院分区：
文献类型：
--
作者：
Brandt, S;Kloska, S;Kehr, J
高通量基因组测序的进步要求开发更有效的方法在细胞水平上检查基因表达。近年来，基于聚合酶链式反应(PCR)的方法已经被开发出来，这种方法可以从少量的材料中扩增mRNA，甚至从单个动物细胞中扩增。同时，几种分析工具可以实现对基因表达的全球监测。到目前为止，高通量分析方法还不能用于单个植物细胞样本。在这里描述的方案中，cDNA阵列杂交(表达谱分析)和使用逆转录酶PCR的扩增策略与来自未受损植物组织的高空间分辨率采样相结合。这一协议为我们提供了一种新的工具，可以在全面的范围内检查组织特异性基因表达模式。为了证明这一工具的有效性，对拟南芥样本中的基因表达模式进行了研究。测定了C24叶片、表皮和叶肉细胞；比较单细胞样本时发现了几个差异表达基因。通过将高通量分析与简单的采样和扩增程序相结合，所述的方法具有提高组织特异性表达分析的效率的潜力。
Advances in high-throughput genome sequencing demand the development of more efficient ways of examining gene expression at a cellular level. During recent years, polymerase chain reaction (PCR)-based methods have been developed that allow the amplification of mRNA from small amounts of material, even from single animal cells. In parallel, several analytical tools permit a global monitoring of gene expression. To date, high throughput analysis methods have not been accessible for single plant cell samples. In the protocol described here, cDNA array hybridization (expression profiling) and an amplification strategy using reverse transcriptase PCR are merged with high spatial resolution sampling from undamaged plant tissue. This protocol gives us a new tool to examine tissue-specific gene expression patterns on a comprehensive scale. To demonstrate the usefulness of this tool, gene expression patterns in samples from Arabidopsis thaliana L. cv. C24 leaf epidermal and mesophyll cells were measured; several differentially expressed genes were identified when single cell samples were compared. The protocol described has the potential of increasing the efficiency of tissue-specific expression analysis by combining high-throughput profiling with straightforward sampling and amplification procedures.