实时RT-PCR定量检测NGF mRNA表达水平方法的建立

Establishment of fluorescent quantitative real time RT-PCR method for detection of the expression of nerve growth factor mRNA

目的:建立一种用SYBR GreenⅠ实时定量逆转录(SYBR GreenⅠQ-RT-PCR)检测大鼠脑组织中神经生长因子(NGF)mRNA基因表达的方法。方法:以Trizol一步法提取大鼠脑组织总RNA,以Oligo(dt)18为引物逆转录产生cDNA,利用SYBRGreenⅠ荧光染料法实时检测NGF mRNA的表达水平。通过熔解曲线分析NGF mRNA检测的特异性,以管家基因β-actin为内参照对NGF进行定量分析。结果:建立的大鼠NGF mRNA SYBR GreenⅠ实时检测方法具有良好的特异性和敏感性。结论:成功建立实时RT-PCR检测NGF mRNA基因表达的方法。

Objective：To establish a new method for detecting the expression of nerve growth factor messenger ribonucleic acid（NGF mRNA） in cerebrum tissue of rats.Methods：Total RNA was isolated from cerebrum tissue by Trizol-one-step method.Specific oligonucleotide of NGF mRNA gene fragments were amplificated by RT-PCR.The method of detection of NGF mRNA expression using SYBR Green Ⅰ real time RT-PCR was constructed.The specificity of the method was confirmed by melting curve.Housekeeping gene β-actin was applied to rectify the quantity of NGF.Results：SYBR Green real-time PCR was developed with high specificity and sensitivity for detecting of rat NGF mRNA.Conclusions：The method of detection of NGF mRNA expression using real time RT-PCR is constructed successfully.