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目的 建立RhD mRNA剪接体实时荧光定量技术,了解不同个体中RhD mRNA剪接体的含量与表达水平。方法 制备本实验室SYBR Green荧光染料定量检测RhD mRNA剪接体的标准曲线,设计RhD mRNA剪接体的引物,提取随机选择的150例血液样品中的mRNA,反转录为cDNA,并以cDNA为模板进行PCR扩增,扩增时加入荧光基团,利用荧光信号实时监控整个PCR进程,最后通过标准曲线来计算RhD mRNA各剪接体的表达量。结果根据荧光定量的峰值读数,以2nd Derivative Max计算方法,计算出所测样本的理论值、实测值,两个重复孔的平均值作为所检测样本RhD mRNA剪接体的表达量。结论 此方法可以准确定量不同个体RhD mRNA剪接体的拷贝数,为RHD血型基因的研究与抗原表达的研究提供依据。
Objective To establish a real-time fluorescence quantitative assay of RhD mRNA splicing and understand the content and expression level of RhD mRNA splicing in different individuals. Methods The standard curve of RhD mRNA splicing was quantitatively detected by SYBR Green fluorescent dye in our laboratory. Primers of RhD mRNA splice body were designed. The mRNA of 150 randomly selected blood samples was extracted and reverse transcribed into cDNA. CDNA was used as template PCR amplification, adding fluorescent groups when amplification, fluorescence signal real-time monitoring of the entire PCR process, and finally by standard curve to calculate the expression of RhD mRNA splicing body. Results According to the peak reading of fluorescence quantitative analysis, the theoretical value and the measured value of the measured sample were calculated by the 2nd Derivative Max calculation method, and the average value of the two duplicate wells was used as the expression level of the RhD mRNA splicing sample. Conclusion This method can accurately quantify the copy number of RhD mRNA splicing in different individuals and provide a basis for the study of RHD blood group genes and antigen expression.