Serial microanalysis of renal transcriptomes
Serial microanalysis of renal transcriptomes
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DOI：
10.1073/pnas.96.26.15286
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发表时间：
1999-12-21
影响因子：
11.1
通讯作者：
Elalouf, JM
中科院分区：
文献类型：
--
作者：
Virlon, B;Cheval, L;Elalouf, JM
大规模的基因表达研究现在可以常规地在大量的细胞上进行，但是目前还不清楚目前的方法在何种程度上对分析复杂组织有价值。在本研究中，我们使用基因表达的系列分析（SAGE）方法对小鼠肾脏中的mRNA进行定量分析。我们首先通过对12，000个mRNA标签进行测序在全肾水平上进行SAGE。最丰富的标签对应于广泛分布或富集在主要肾上皮细胞（近端肾小管细胞）中的转录物，而对次要细胞类型特异性的转录物几乎没有得到证明。为了更好地探索这些细胞，我们建立了用于缩小提取物的SAGE适应，使起始材料的量减少1,000倍。通过研究显微切割的肾小管（50，000个细胞）中的基因表达来评估这种方法的潜力。获得了特异性基因表达谱，并且已知的标记物（例如，在Henle袢的粗升支中的尿调节蛋白和在集合管中的水通道蛋白-2）被发现适当地富集。此外，几个丰富的标签没有数据库匹配，这表明他们对应于未知的或不好的特点与特定的组织分布的转录本，它的结论是，SAGE适应缩小提取物使得可能大规模的定量基因表达测量在小的生物样品，并将有助于研究组织表达和功能的基因没有证明与其他高通量的方法。
Large-scale gene expression studies can now he routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues, In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced, To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution, It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.