Microscale cDNA subtraction method for detection of regulators for maxillo-facial development.

消减杂交方法用于检测在特定组织或细胞中表达而在另一组织或细胞中不表达的基因。通常，含有差异表达基因的称为“测试者”的cDNA与不含此类基因的称为“驱动者”的过量RNA杂交。结果，差异表达的cDNA富集在未杂交的cDNA组分中。传统的消减杂交方法虽然在某些情况下得到了成功的应用，但需要大量的测试者和驱动者mRNA，因此，当只需要少量的组织（如小鼠牙芽等）时，很难鉴定出差异表达的基因。本研究采用两种不同的方法构建了软骨细胞特异性消减cDNA文库：[1]常规单链消减法（tester cDNA-driver mRNA）和[2]双链消减法（tester dscDNA-driver dscDNA）。在这两种方法中，我们使用PCR扩增测试者cDNA。我们发现这些文库的质量相似，但在方法[2]中构建文库所需的测试者mRNA的量比[1]少得多。在两个文库中，大部分cDNA克隆编码3'端非翻译序列和部分序列，插入的cDNA平均大小约为550 bp。我们推测这是由于PCR扩增的限制。虽然部分cDNA可以作为探针来检测基因在组织中的表达模式，但需要对未消减文库进行筛选以获得全长cDNA。

Subtractive hybridization methods are used to detect genes that are expressed in a specific tissue or cell but not in the other. Usually cDNA called "tester" that contains differentially expressed genes is hybridized with an excess amount of RNA called "driver" that does not contain such genes. As a result, differentially expressed cDNA in enriched in unhybridized cDNA fraction. Although traditional subtractive hybridization methods have been successfully used in some cases, they require a lot of tester and driver mRNA.Therefore, it has been difficult to identify differentially expressed genes when only small amount of tissue (e.g.mouse tooth bud etc.) is available as a starting material.In the present study, we constructed chondrocytes specific subtraction cDNA library by two different methods : [1] conventional single strand subtraction (tester cDNA-driver mRNA) and [2] double strand cDNA subtraction (tester dscDNA-driver dscDNA). In both methods, we used PCR for amplifying tester cDNA.We found the quality of those libraries was similar but the amount of tester mRNA that was required for library construction was much smaller in the method [2] than that of [1]. In both libraries, most of cDNA clones encodes 3' untranslated sequence plus partial sequence and the average size of inserted cDNAs was about 550bp. We speculated this was due to the limitation of PCR amplification. Although partial cDNA may be useful as a probe to examine expression pattern of the gene in the tissue, It is necessary screen the unsubtracted library to obtain full length cDNA.