Quantification of thromboxan A_2 receptor mRNA and prostacyclin receptor mRNA in human platelet

建立了一种简便、快速、可靠的定量检测微量mRNA的酶免疫分析方法，并将其应用于人血小板血栓素A_2受体(TXA_2R)和前列环素受体(PGI_2R)mRNA的检测。在逆转录聚合酶链式反应过程中，用已经过生物化的引物对其进行标记。通过碳化二亚胺反应将长约20个LIP的寡核苷酸探针固定在包被的赖氨酸表面，碱变性后，DNA片段在微滴定孔板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聚合酶链式反应-Southern法进行了比较。血栓素A_2R和前列环素受体的最低检测浓度分别为50pg/50MU_1和5pg/50MU_1，而溴化依利地铵显色法和聚合酶链式反应-Southern法的最低检测浓度分别为600pg/50MU_1和500，50pg/50MU_1。20名健康志愿者的血栓素A_2R和前列环素_2R基因分别为274.4和386.9255.6pg/50MU_1。

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.