Quantification of thromboxan A_2 receptor mRNA and prostacyclin receptor mRNA in human platelet
人血小板中血栓素 A_2 受体 mRNA 和前列环素受体 mRNA 的定量
基本信息
- 批准号:09672366
- 负责人:
- 金额:$ 0.9万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:1997
- 资助国家:日本
- 起止时间:1997 至 1998
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
本文建立了一种简便、快速、可靠的定量微量mRNA的酶免疫分析法(EIA),并应用于人血小板血栓素A_2受体(TXA_2R)mRNA和前列环素受体(PGI_2R)mRNA的测定。在RT-PCR的过程中,使用已经生物素化的引物标记cDNA片段。用碳二亚胺法将长度约20 bp的寡核苷酸探针通过其5 '端的磷酸基团固定在赖氨酸包被的孔表面,经碱变性后,在微量滴定板的威尔斯孔上与寡核苷酸探针杂交。用链霉亲和素标记的过氧化物酶测定杂交DNA的浓度,并与溴化乙锭染色法和PCR Southern法进行比较。微量滴定法检测TXA_2RmRNA的最低浓度为50 pg/50 μ l,PGI_2RmRNA的最低浓度为5 pg/50 μ l,溴化乙锭染色法和PCR-Southern法的最低浓度分别为600,100 pg/50 μ l和500,50 pg/50 μ l。血小板TXA_2RmRNA和PGI_2RmRNA分别为274.4 ± 214.9pg/50 μ l和386.9255.6pg/50 μ l。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ANDO Yasuhiko其他文献
ANDO Yasuhiko的其他文献
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{{ truncateString('ANDO Yasuhiko', 18)}}的其他基金
Fundamental and clinical studies of flow Cytometric analysis of activated plateles using Activation in Patients with Sleep Apnea Syndrome
使用 Activation 对睡眠呼吸暂停综合征患者的活化血小板进行流式细胞术分析的基础和临床研究
- 批准号:
11672309 - 财政年份:1999
- 资助金额:
$ 0.9万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Platelet prostacyclin receptors in myocardial infarction
心肌梗死中的血小板前列环素受体
- 批准号:
04671441 - 财政年份:1992
- 资助金额:
$ 0.9万 - 项目类别:
Grant-in-Aid for General Scientific Research (C)
Studies on PGE_1/PGI_2 receptors in platelets from patients with thrombotic disorders
血栓性疾病患者血小板中PGE_1/PGI_2受体的研究
- 批准号:
63571113 - 财政年份:1988
- 资助金额:
$ 0.9万 - 项目类别:
Grant-in-Aid for General Scientific Research (C)