竞争性RT-PCR测定法及BMP-2mRNA的定量检测

A Competitive RT-PCR Assay and Quantitation of BMP-2 mRNA

建立竞争性 PCR方法 ,以期用于转录水平基因表达的定量研究 .以检测大鼠颅骨骨细胞总RNA中骨形态发生蛋白 - 2 ( BMP- 2 ,bone morphogenetic protein- 2 ) m RNA含量为例 ,构建大鼠BMP- 2基因的竞争模板 ,以之为内对照进行竞争性 PCR.PCR反应结束后 ,电泳、拍照 ,扫描所扩增条带密度 ,作回归方程 .根据回归方程 ,计算出正常 7d Wistar大鼠颅骨总 RNA中 BMP- 2 m RNA含量为 1 .1 2 5amol/μg RNA.结果显示 ,成功构建了大鼠 BMP- 2基因的竞争模板 ,建立了可以测定大鼠颅骨骨细胞总 RNA中 BMP- 2 m RNA含量的竞争性 PCR方法 .

In order to study the regulation of gene expression and to acquire quantitative information on mRNA of an individual gene,a competitive PCR was established.A competitor of rat BMP 2 (bone morphogenetic protein\|2) gene was constructed.BMP\|2 primer sequences were added to an Eco RⅠ\| Bam H I fragment of V\|erbB oncogene,so the fragment had the same primer sets with BMP 2.Then the competitor and the cDNA that was generated from BMP 2 mRNA by reverse transcription were co\|amplified in one PCR tube,and the products were analyzed by gel electrophoresis.Intensities of the competitor and BMP 2 gene bands were determined by scanning the photo film with a computer scanner.A linear regression equation was established based on data of lg(BMP 2/competitor) and the amounts of the competitor.According to the linear regression equation,the amount of BMP 2 mRNA in total calvarial RNA of normal 7\|days Wistar rats was determined,which was 1.125 amol/μg RNA.The results showed that a competitive PCR had been successfully developed for quantification of the amount of BMP 2 mRNA in total calvarial RNA and satisfied results obtained.