实时荧光定量聚合酶链反应突触后密度蛋白93基因TaqMan探针的制备

目的:制备实时荧光定量聚合酶链反应(FQ-PCR)突触后密度蛋白93(PSD93)基因TaqM an探针以进一步研究PSD93表达情况。方法:采用RT-PCR法,从生后1天的SD大鼠大脑组织mRNA中扩增PSD93基因部分CDS区片段,克隆入T载体,筛选阳性克隆、酶切鉴定及序列测定,采用TaqM an探针,以重组质粒为模板绘制标准曲线。结果:RT-PCR法扩增出一特异产物与预期长度113bp相符,DNA序列测序的结果与GenBank提供的已知序列(RNU50717)比较,所克隆的PSD93基因片段与其中的113bp完全相同,与PSD93基因100%同源。以重组质粒为模板绘制标准曲线,相关系数r大于0.99,反应效率为0.95,各点变异系数小于20%。结论:采用RT-PCR和T载体技术成功获得FQ-PCR PSD93基因TaqM an探针。

The preparation of PSD93 TaqMan probe for real - time fluorescence quantitative PCR

Objective:To obtain the probe of postsynaptic density 93(PSD93) for real-time fluorescence quantitative PCR(FQ-PCR).Methods:The partial CDS of PSD93 was amplified by RT-PCR from 1d postnatal SD rat brain.RT-PCR product was ligated into pGEM-T vector,and the DNA sequence was detected.The standard curve was drawn with the recombinant plasmids and the PSD93 TaqMan probe.Results:The product of RT-PCR was 113 bp which matched the size of the purpose.This DNA sequence was 100% homogeneous with the PSD93 cDNA reported. The correlation coefficient of the standard curve was above 0.99,the reaction efficiency was 0.95 and all the variation coefficients were below 20%.Conclusion:The PSD93 TaqMan probe for FQ-PCR has been successfully obtained with RT-PCR and T vector techniques.