蒙古绵羊输卵管上皮细胞β-防御素mRNA相对表达水平的实时荧光定量PCR检测

Real-time PCR detection of relative expression of sBD-1 mRNA of epithelial cell of fallopian tubes in Mongolian sheep

为了检测培养的蒙古绵羊输卵管上皮细胞内sBD-1 mRNA表达水平,运用SYBR GreenⅠ实时荧光定量PCR技术进行了相对定量测定。首先,建立蒙古绵羊输卵管上皮细胞培养体系作为体外试验模型,提取细胞总RNA,根据GenBank中羊β-防御素基因序列,设计合成引物,进行实时荧光定量PCR。以β-Actin基因为内参基因,对sBD-1 mRNA进行均一化处理,利用荧光阈值(Ct值)计算sBD-1 mRNA的相对表达量。经测序分析,扩增产物为β-防御素。结果显示,SYBR GreenⅠ实时荧光定量PCR技术可以测定蒙古绵羊β-防御素mRNA表达水平的相对含量。

To detect the relative expression of sBD-1 mRNA in the cultured epithelial cell of fallopian tubes of Mongolian sheep,a real-time PCR with SYBR GreenⅠ was performed.The in vitro model of cultured system of fallopian tubes epithelial cell in Mongolian sheep was established,and the total RNA of epithelial cell was extracted.With β-Actin gene as a reference gene,a real-time PCR with SYBR GreenⅠ was carried out.The amplified product was confirmed to be β-defensins gene by sequencing.The results showed that the relative expression of sBD-1-mRNA in the cultured epithelial cells of Mongolian sheep could be detected using the real-time PCR with SYBR GreenⅠ.