【摘要】 目的:建立准确定量血清中hsa_circRNA₁03191的绝对反转录荧光定量PCR（RT-qPCR）方法,并在肝癌患者外周血清中验证hsa_circRNA₁03191的拷贝数。方法:提取肝癌患者肿瘤组织和血清中的总RNA,反转录获得cDNA,再以cDNA为模板,PCR法扩增出包含反向剪接位点的hsa_circ RNA₁03191的119bp特异性片段,克隆到T载体中,经蓝白斑筛选,测序鉴定重组质粒。对重组质粒进行梯度稀释成标准品。采用SYBR荧光染料法进行扩增,建立标准曲线,在肝癌患者外周血中验证hsa_circRNA₁03191的copies。结果:肝癌患者外周血清中游离的总RNA较肝癌组织中总RNA降低（t=10.95,p<0.0001）。建立了能准确定量血清中GAPDH和hsa_circRNA₁03191拷贝数的RT-qPCR方法,使用该定量方法能检测血清中GAPDH和hsa_circRNA₁03191的表达。结论:建立了检测hsa_circRNA₁03191的绝对荧光定量RT-qPCR方法,该法检测线性范围广,灵敏度、特异性、重复性均良好,可为血清中circRNA精确定量提供参考方法。更多还原

【Abstract】 Objective: to establish an absolute reverse transcription fluorescence quantitative PCR method（RT-qPCR） that can accurately quantify hsa_circRNA 103191 in serum, and verify the copy number of hsa_circRNA₁03191 in peripheral blood serum of patients with primary hepatic carcinoma（PHC）. Methods: Total RNA was extracted from tumor tissue and serum of patients with PHC,and cDNA was abtained by reverse transcription. The 119 bp specific fragment containing the back-spliced junction of hsa_circRNA₁03191 was amplified by PCR using cDNA as template, and cloned into T vector, and then the recombinant plasmid was sequenced after blue and white spot screening. Using the gradient diluted recombinant plasmid as standard substance, the standard curve of hsa_circRNA 103191 was established by RT-qPCR method. At last the copy number of hsa_circRNA 103191 in peripheral blood of patients with PHC was verified. Results: The free total RNA in peripheral blood of patients with PHC was lower than that in PHC tissues（t=10.95,P<0.0001）.The absolute RT-qPCR method was established that can accurately quantify the copy number of GAPDH and hsa_circRNA₁03191.The expression of GAPDH and hsa_circRNA₁03191 in serum can be detected by this method. Conclusion: The absolute RT-qPCR method for the detection of hsa_circRNA₁03191 was established and had a wide linear range, high sensitivity and specificity, and good repeatability, and could be used as a reference method for the accurate quantification of circRNAs in serum.更多还原