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

本文建立了一种简便、快速、可靠的微量mRNA定量酶免疫分析法（EIA），并将其应用于人血小板中血栓素A_2受体（TXA_2R） mRNA和前列腺素受体（PGI_2R） mRNA的定量分析。该方法是利用固定在微滴板固体表面的寡聚探针，用RT-PCR扩增cDNA片段进行简单杂交。在RT-PCR过程中，cDNA片段用已经被生物素化的引物进行标记。长度约为20唇的寡聚探针通过其5′端磷酸基团通过碳二亚胺反应将其固定在赖氨酸表面。碱液变性后，DNA片段在微滴板孔上与寡聚探针杂交。杂交DNA的浓度由链霉亲和素偶联过氧化物酶测定。将该方法与溴化乙锭染色法和PCR Southern法进行比较。微量滴度法检测到的TXA_2R mRNA最低浓度为50 pg/50 μ 11， pgi2r mRNA最低浓度为5 pg/50 μ 1。相反，溴化乙啶染色法和PCR Southern法分别为600，lOOpg/50 mu1和500,50 pg/50 mu1。在20名正常志愿者中，血小板TXA2R mRNA和pgi2r mRNA分别为274.4 214.9 pg/50 mu1（平均值）和386.9255.6 pg/50 mu1。

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.