FQ-PCR检测小鼠肝癌模型中Cyclin E mRNA方法的建立

目的:建立SYBR Green Ⅰ实时荧光PCR定量检测小鼠肝癌模型中细胞周期蛋白cyclin E mRNA的方法。方法:以cyclin E和β-actin的PCR产物为阳性模板,分别将二者cDNA产物进行10倍梯度稀释,建立双标准曲线,应用SYBR Green Ⅰ实时荧光定量PCR检测cyclin E和β-actin mRNA含量,对熔解曲线进行分析确定扩增产物的特异性。结果:建立的标准曲线线性关系良好,相关系数r均为-1.00,cyclin E和β-ac-tin扩增效率分别为0.91和0.93,熔解曲线分析显示均为单峰,特异扩增产物的Tm值分别为(81.84±0.28)℃和(89.15±0.26)℃。10-4Cyclin E和β-actin cDNA标本日间变异系数(CV)和批内CV分别为1.02%、1.45%和0.90%、1.48%。结论:SYBR Green I实时荧光PCR定量检测cyclin E mRNA是一种方便快捷、经济实惠、灵敏度高、特异性好的方法,为cyclin E的进一步研究奠定方法学基础。

Development of a real-time fluovescent PCR assay for detecting cyclin E in mouse models of hepatocarcinoma

Objective: To establish a real-time fluorescent polymerase chain reaction (PCR) products of cyclin E and β-actin,The mRNA expressions of them were analyzed quantitatively by real-time fluorescent polymerase chain reaction(FQ-PCR) with SYBR Green I,and the characteristic of specific cyclin E and β-actin amplicon was analyzed by melting curve. Methods: Standard curve was established by using a 10-fold series dilution of the PCR products of cyclin E and β-actin,The mRNA expressions of them were analyzed quantitatively by real-time fluorescent polymerase chain reaction(FQ-PCR) with SYBR Green I,and the characteristic of specific cyclin E and β-actin amplicon was analyzed by melting curve. Results: The results showed that two standard curves estab-lished had good linear dependence and the correlation coefficient was-1.00,PCR efficiency of cyclin E and β-actin is 0.91 and 0.93,respectively.The melting curve analysis showed the product was specific to a single peak,and Tm was(81.84±0.28)℃ and(89.15±0.26)℃ respectively.The within-run and between-day coefficient of variability(CV) of 10-2 cDNA of cyclin E and β-actin was 1.02%,1.45% and 0.90%,1.48% respectively. Conclusion: SYBR Green I real-time fluorescent quantitative polymerase chain reaction is a rapid,economics,sensitive,and specific method,it provided useful methodological basis for further research on cyclin E.