Real time RT-PCR定量检测BDNF mRNA表达水平方法的建立

Establishment of A Real Time Method for Detecting the Expression of BDNF mRNA Gene

目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.

Objective To establish a real time RT -PCR method for detecting the expression of BDNF mRNA （brain -derived neurotrophic factor messenger ribonucleic acid, BDNF mRNA） in cerebrum tissue after MCAO/R （middle cerebral artery occlusion reperfusion, MCAO/R） injury in rats. Methods Total RNA was isolated from cerebrum tissue. Specific oligonucleotide of BDNF mRNA gene fragments were amplified by RT - PCR. The products of RT - PCR were recombined to plasmid and sequenced. The method of detecting BDNF mRNA expression by real time RT - PCR was established. Results The recombinant plasmid was digested by enzymes and sequenced. It was proved that the specific fragment had been cloned to vectors. The kinetics curves reflecting the progress of gene amplification and a quantitative standard curve were completed. Conclusions The method of detecting BDNF mRNA expression by real time RT - PCR is constructed successfully.