摘要
目的 建立realtime逆转录聚合酶链反应 (RT PCR)检测AFPmRNA表达水平方法。方法 提取BEL740 2细胞总RNA ,进行RT PCR扩增并纯化AFP基因片段 ,构建重组质粒 ,建立realtimeRT PCR检测AFPmRNA表达水平方法。结果 重组质粒的阳性克隆效率为 81.8% ,经酶切鉴定 ,目的基因片段已插入PMD 18T载体内 ,得到realtimeRT PCR动力学曲线。结论 成功建立了realtimeRT PCR定量检测AFPmRNA表达水平方法。
Objective To construct the method of detection of AFP(alpha fetoprotein) mRNA expression using real time reverse transcriptional polymerase chain reaction(RT PCR). Methods Total mRNA of BEL7402 cells was extracted. The RT PCR products of AFP mRNA were purified and combined with PMD 18T vectors. The method of detection of AFP mRNA expression using real time RT PCR were constructed.Results The positive efficiency of recombinant plasmids were 81.8%. It was proved the aimed gene had been cloned into PMD 18T vectors by enzymes digesting. The kinetics curves reflecting the progress of gene amplification and a quantitative standard curve were completed.Conclusion The method of detection of AFP mRNA expression using real time RT PCR is constructed successfully.
出处
《新乡医学院学报》
CAS
2003年第5期308-309,313,共3页
Journal of Xinxiang Medical University
基金
20 0 2年河南省科技厅发展计划项目(0 2 2 463 0 0 3 2 )
2 0 0 3年河南省教育厅自然科学研究项目资助(2 0 0 3 3 2 0 14 6)
关键词
甲胎蛋白
mRNA定量
逆转录聚合酶链反应
alpha fetoprotein
quantitation of mRNA
reverse transcriptional polymerase chain reaction
