| Budget Amount *help |
¥3,810,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥210,000)
Fiscal Year 2007: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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
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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
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