BMPR-Ⅱ基因mRNA荧光定量PCR检测标准品的构建

Construction of the standards for detecting human BMPR-Ⅱ mRNA with real-time fluorescence quantitative polymerase chain reaction

目的构建检测BMPR-Ⅱ基因mRNA表达水平差异的标准品。方法以人胚肺成纤维细胞的总RNA为模板、O ligo(dT)18为引物逆转录产生cDNA,用该cDNA为模板PCR扩增人BMPR-Ⅱ基因相应的cDNA片断,构建pMD-18-BMPR-Ⅱ重组质粒,经鉴定测序,用荧光定量PCR制作标准曲线。结果成功克隆了人BMPR-ⅡcDNA,并以其为标准制作出荧光定量PCR标准曲线。结论成功构建了BMPR-Ⅱ基因荧光定量PCR标准质粒,为今后BMPR-ⅡmRNA的荧光定量PCR检测打下了基础。

Objective To Clone human BMPR-Ⅱ cDNA and using it as the standard for real-time quantifying BMPR-Ⅱ mRNA. Methods Total RNA was extracted from the Human embryonic lung fibroblast cells and reverse transcribed to cDNA using Oligo （dT） 18 as primer. Amplified BMPR-Ⅱ cDNA was ligated with pMD 18-T simple vector and transformed to bacteria JM109. Plasmids were extracted from positive clones and identified with PCR, and then were subjected to sequencing. At last we make a standard for real-time PCR analysis. Results The human BMPR-Ⅱ cDNA fragment had been cloned successfully, the standard curve was made for detecting human BMPR-Ⅱ mRNA with real-time fluorescence quantitative polymerase chain reaction. Conclusion The recombined plasmids containing human BMPR- Ⅱ cDNA have been constructed successfully; it established the foundation for the detection of the BMPR- Ⅱ mRNA with the real-time PCR.