血清中hsa_circRNA_103191绝对荧光定量PCR方法的构建及应用

Establishment and application of an absolute fluorescence quantitative PCR method for hsa_circRNA_103191 in serum

目的:建立准确定量血清中hsa_circRNA_103191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_circRNA_103191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 RNA_103191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_circRNA_103191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_circRNA_103191拷贝数的RT-qPCR方法,使用该定量方法能检测血清中GAPDH和hsa_circRNA_103191的表达。结论:建立了检测hsa_circRNA_103191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。

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_circRNA_103191 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 119bp specific fragment containing the back-spliced junction of hsa_circRNA_103191 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_103191was 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_circRNA_103191.The expression of GAPDH and hsa_circRNA_103191 in serum can be detected by this method.Conclusion:The absolute RT-qPCR method for the detection of hsa_circRNA_103191 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.