hsa＿circ＿0004123绝对定量PCR方法的建立及验证

Establishment and validation of hsa＿circ＿0004123 absolute quantitative PCR method

目的建立能精确定量hsa_circ_0004123的绝对反转录荧光定量PCR(RT-qPCR)方法,并在白血病细胞系中验证hsa_circ_0004123的拷贝数。方法提取B淋巴母细胞系HMy2.CIR总RNA,反转录合成cDNA。以cDNA为模板,扩增包含hsa_circ_0004123反向剪接位点的236 bp特征片段,克隆到pGM-T载体中,并对重组质粒进行测序鉴定。以梯度稀释的重组质粒为标准品,采用SYBR荧光染料法进行RT-qPCR并建立标准曲线,在4种白血病细胞系(HMy2.CIR、Jurkat、Raji、K562)中验证hsa_circ_0004123的拷贝数。结果建立了能精确定量hsa_circ_0004123拷贝数的绝对RT-qPCR方法,其线性范围为(9.63×10^3~9.63×10^9)拷贝/μl,灵敏度为(4528±516)拷贝/μl,批内变异系数(CV)为0.30%~1.86%,批间CV为1.40%~2.30%,且熔解曲线为单一峰值曲线。对4种白血病细胞系(HMy2.CIR、Jurkat、Raji、K562)中hsa_circ_0004123的拷贝数分别用本法进行检测,结果显示,hsa_circ_0004123在4种白血病细胞系中的表达差异有统计学意义(P<0.001)。结论建立的检测hsa_circ_0004123的绝对RT-qPCR方法具有线性范围广,灵敏度、特异性高,重复性好等特点,可为hsa_circ_0004123在白血病中的精确定量提供技术支持。

Objective To establish an absolute reverse transcription fluorescence quantitative PCR method(RT-qPCR)that can accurately quantify the circRNA hsacirc0004123,and verify the copy number of hsacirc0004123 in the leukemia cell line.Methods Total RNA was extracted from HMy2.CIR cell line,and reverse transcribed to cDNA.Using cDNA as template,a 236 bp characteristic segment containing the back-spliced junction of hsacirc0004123 transcript was cloned and inserted into the pGM-T vector,and then the recombinant plasmid was sequenced.Regarding the gradient diluted recombinant plasmid as standard substance,the standard curve of hsacirc0004123 was established by SYBR fluorescent staining RT-qPCR,and then the copy number of hsacirc0004123 in leukemia cell lines(HMy2.CIR,Jurkat,Raji,K562)were verified.Results The absolute RT-qPCR method was established that can accurately quantify the copy number of hsacirc0004123 with wide linear range(9.63×10^3–9.63×10^9)copies/μl,high sensitivity(4528±516)copies/μl,intra-batch coefficient of variation(CV)of 0.30%-1.86%,inter-batch CV of 1.40%-2.30%,and a single peak dissolution curve.The copy number of hsacirc0004123 in leukemia cell lines HMy2.CIR,Jurkat,Raji and K562 was detected respectively.The expression of hsacirc0004123 was significantly different(P<0.001)in the 4 kinds of leukemia cell lines.Conclusion The absolute RT-qPCR method used to detect the copy number of hsacirc0004123 has a wide linear range,high sensitivity and specificity,and good repeatability,and lays a technical foundation for accurate quantification of hsacirc0004123 in leukemia.