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Development of cDNA array for diagnosis of sarcoma and its application to routine pathological diagnosis

肉瘤诊断cDNA芯片的研制及其在常规病理诊断中的应用

基本信息

批准号：
16590269
负责人：
TAKAZAWA Yutaka
金额：
$ 2.44万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16590269/
关键词：
sarcoma chimeric gene patholoeical diagnosis cDNA array キメラ遣伝子

项目摘要

Sarcoma is classified into numerous subtypes, reflecting the variety of mesenchymal tissue firm which they are originated. For this reason Pathological diagnosis of sarcoma is often difficult, combined with rarity of each subtype, despite many highly malignant subtypes are comprised in sarcoma On the other hand, correct pathological diagnosis is indispensable far appropriate clinical management of sarcoma Recently, reciprocal chromosomal translocation and formation of chimeric gene, which are highly specific to individual sarcoma subtypes, were found in many tumors. In order to improve the accuracy of pathological diagnosis of sarcoma, we developed a new method for molecular detection of chimeric genes using DIN array technology RNA was extracted from synovial sarcoma, clear cell sarcoma and Ewing sarcoma, followed by conversion to cDNA Each chimeric gene was amplified by multiplex PCR with mixture of primers of various chimeric genes, at the same time, SP6 promoter was inserted to PCR … More product Biotin-labeled cRNA was generated them PCR products by in vitro transcription using SP6 promoter; then it was hybridized to macroarray slides, a membrane-attached glass slides on which the probes corresponding to exons of each chimeric gene were spotted. Positive signals were visualized by alkaline phosphatase.By this multiplex PCR-cDNA array method, correct chimeric genes and breakpoints were detected in most tumors. These results were validated by direct sequencing of PCR products of chimeric genes. Moreover, this method was applicable to routine pathological laboratory, because each procedure was simple and no special equipments was required except for a PCR machine. All the procedures are to be finished in about 3 days and resulted macroarray slides are able to store with other histopathological slides.As for diagnostic aspects, this method might improve accuracy of pathological diagnosis of sarcoma, because it enables molecular pathological diagnosis independent from histopathological diagnosis. Less

肉瘤分为许多亚型，反映了它们起源于间充质组织的多样性。因此，肉瘤的病理诊断往往很困难，再加上各亚型的少见，尽管肉瘤中包含许多高度恶性的亚型，但正确的病理诊断对于肉瘤的临床治疗是必不可少的。近年来，在许多肿瘤中发现了高度特异的染色体相互易位和嵌合基因的形成。为了提高肉瘤病理诊断的准确性，我们建立了一种利用DIN芯片技术进行嵌合基因分子检测的新方法，从滑膜肉瘤、透明细胞肉瘤和尤文肉瘤中提取rna，然后用不同嵌合基因的引物进行多重聚合酶链式反应，扩增出每个嵌合基因，同时将sp6启动子插入到聚合酶链式反应…用SP6启动子体外转录产生更多的生物素标记的cRNA，然后与大芯片玻片杂交，大阵列玻片是一种贴膜的玻片，在其上可以看到每个嵌合基因外显子对应的探针。用碱性磷酸酶显示阳性信号，通过这种多重PCR-cDNA阵列方法，在大多数肿瘤中检测到正确的嵌合基因和断裂点。通过对嵌合基因的聚合酶链式反应产物直接测序，验证了这些结果。此外，该方法还适用于常规病理实验室，因为每个步骤都很简单，除了一台PCR机外，不需要特殊的设备。在诊断方面，这种方法可以提高肉瘤病理诊断的准确性，因为它使分子病理学诊断独立于组织病理学诊断。较少