Datson N A, van der Perk-de Jong J, van den Berg M P, de Kloet E R, Vreugdenhil E
Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands.
Nucleic Acids Res. 1999 Mar 1;27(5):1300-7. doi: 10.1093/nar/27.5.1300.
Serial Analysis of Gene Expression (SAGE) is a powerful expression profiling method, allowing the analysis of the expression of thousands of transcripts simultaneously. A disadvantage of the method, however, is the relatively high amount of input RNA required. Consequently, SAGE cannot be used for the generation of expression profiles when RNA is limited, i.e. in small biological samples such as tissue biopsies or microdissected material. Here we describe a modification of SAGE, named microSAGE, which requires 500- to 5000-fold less starting material. Compared with SAGE, microSAGE is simplified due to incorporation of a 'single-tube' procedure for all steps from RNA isolation to tag release. Furthermore, a limited number of additional PCR cycles are performed. Using microSAGE gene expression profiles can be obtained from minute quantities of tissue such as a single hippocampal punch from a rat brain slice of 325 micrometers thickness, estimated to contain, at most, 10(5) cells. This method opens up a multitude of new possibilities for the application of SAGE, for example the characterization of expression profiles in tissue biopsies, tumor metastases or in other cases where tissue is scarce and the generation of region-specific expression profiles of complex heterogeneous tissues.
基因表达序列分析（SAGE）是一种强大的表达谱分析方法，可同时分析数千个转录本的表达情况。然而，该方法的一个缺点是所需的输入RNA量相对较高。因此，当RNA有限时，即对于诸如组织活检或显微切割材料等小生物样本，SAGE不能用于生成表达谱。在此，我们描述了一种SAGE的改进方法，称为微量SAGE（microSAGE），它所需的起始材料减少了500至5000倍。与SAGE相比，由于从RNA分离到标签释放的所有步骤都采用了“单管”程序，microSAGE得到了简化。此外，还进行了有限数量的额外PCR循环。使用microSAGE，可以从微量组织中获得基因表达谱，例如从厚度为325微米的大鼠脑切片上取下的单个海马打孔组织，估计该组织最多包含10⁵个细胞。这种方法为SAGE的应用开辟了许多新的可能性，例如在组织活检、肿瘤转移或其他组织稀缺的情况下表征表达谱，以及生成复杂异质组织的区域特异性表达谱。
