| Project/Area Number |
09672366
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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 |
Laboratory medicine
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| Research Institution | Tokai University |
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Principal Investigator |
ANDO Yasuhiko School of Medicine, Tokai University Department of Clinical Pathology Professor, 医学部, 教授 (50051470)
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| Co-Investigator(Kenkyū-buntansha) |
宮地 勇人 東海大学, 医学部, 助教授 (20174196)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Thromboxane A_2 receptor (TXA2R) mRNA / prostacyclin receptor (PGI2R) mRNA / Lysinplate hybridization assay / Bhidiumbromide stain / PCR Southern method / ELISA assay / プロスタサイクリンレセプター(以下PGI_2R)mRNA / ヒト血小板 / PGI_2レセプター / TXA_2レセプター / m-RNA / RT-PCR / ビオチン化プライマー / 定量化 / ELISA系アッセイ |
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| Research Abstract |
We developed a simple, rapid and reliable enzyme immunoassay(EIA) to quantify minute amount of mRNA, and applied it for the assay of thromboxan A_2 receptor (TXA_2R) mRNA and prostacyclin receptor(PGI_2R) mRNA in human platelets The assay is based on simple hybridization method for a cDNA segment which is amplified by the RT-PCR from mRNA with oligoprobe which is immobilized on the solid surface of microtiter plate well. in the process of RT-PCR, the cDNA segments are labeled by using primer which is already biotinated. The oligoprobe with a length of about 20 lip is immobilized via its 5'-terminal phosphate groups to the surface of lysine coated well by carbodiimide reaction.
After alkali denaturation, the DNA segment is hyblidized with the oligoprobe on microtiter plate wells . The concentration of the hybridized DNA is determined by streptavidin-conjugated peroxidase.
This microtiter method was compared with ethidium bromide staining method and PCR Southern method for mRNA detection. The lowest concentrations that were detectable by the microtiter method was 50 pg/50mu 11 for TXA_2R mRNA and 5 pg/50 mu 1 for PGI_2R mRNA.On the contrary, those of etliidium bromide staining method and PCR Southern method were 600, lOOpg/50 mu1 and 500, 50 pg/50 mu1, respectively.
In 20 normal volunteers, TXA2R mRNA and PGI_2R mRNA in platelets were 274.4 214.9 pg/50 mu1 (meanSD) and 386.9255.6 pg/50 mu1.
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