2004 Fiscal Year Final Research Report Summary
Microarray and microdissection for analysis of mechanisn on human chronic allograft nephropathy
| Project/Area Number |
15591708
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | Osaka City University |
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Principal Investigator |
UCHIDA Jung Osaka City University, Graduate School of medicine Urology, Research Associate, 大学院・医学研究科, 助手 (40343412)
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| Co-Investigator(Kenkyū-buntansha) |
NAKATANI Tatsuya Osaka City University, Graduate School of medicine Urology, Professor, 大学院・医学研究科, 教授 (40183511)
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| Project Period (FY) |
2003 – 2004
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| Keywords | DNA tip / Chronic allograft nephropaty / Acute resection / cDNA microarray / microdissection |
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| Research Abstract |
To diagnose acute rejection induced chronic allograft nephropathy in the future and to judge the efficacy of treatment for rejection is often difficult. It is expected to detect the specific marker for acute rejection. We used DNA microarrays in a systematic study of gene-expression profile in tubulointerstitunum obtained by laser-captured microdissection in frozen biopsy samples from patients with acute rejection.
Biopsy samples from patients with acute rejection (n=3) and control (n=3) were embedded in Tissue Tek OCT medium and snap-frozen. The cryostat section (8μm) were made, and tubulointestinum was laser microdissected with LM-2, Arcturus Engineering. Total RNA was extracted from dissected tissues. RNA was amplified by a T7 RNA polymerase-mediated RNA amplification reaction combined with an adaptor ligation-mediated PCR (TALPAT method). After biotinylated cRNAs were fragmented, they were hybridized to the DNA tip (Human genome U133 Plus 2.0 array : 54675 genes) and signal intensity was measured by Affymetrix gene Tip system. Data analysis was performed with Gene Spring. After eliminating data showing mismatch hybridization with negligible expression in both acute rejection and control group, 18356 genes were detected. Two hundred fifty three genes were twice or more differences in expression between acute rejection group and control group.
We exploited gene tip technology to identify genes expression in the specific region where acute rejection occurred by biopsy samples from patients with acute rejection.
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