实时荧光定量PCR筛选牛源整联蛋白αv基因的siRNA片段

Selection of siRNA Fragment of Cattle-borne Integrin-αv Gene by Real-time Fluorescence Quantitative PCR

设计并合成三条针对牛口蹄疫病毒(FMDV)整联蛋白受体αv亚基基因的特异性干扰小RNA(SiRNA)片段,将其转染至本身表达整联蛋白受体αv亚基基因的MDBK细胞。分别在转染后36 h和48 h收集细胞,提取细胞总RNA并反转录为cDNA。应用SYBR GreenⅠ实时荧光定量RT-PCR的方法检测3个siRNA片段的干扰效果,筛选出对整联蛋白αv基因具有高效干扰效果的小RNA片段。为建立抗口蹄疫病毒牛新品种培育的RNA干扰转基因技术平台奠定了基础。

In order to select the most efficient small interference RNA among three RNA oligoes which aim at downregulateing the expression of integrin-αv,the three RNA oligoes were transfected into the MDBK cells which express the integrin-αv gene.The cells were harvested in 36 hours and 48 hours respectively after transfection,and then the total RNA from these cells was extracted.After transcripting the RNA into cDNA by RT-PCR,these cDNA as templates carrying out fluorescence quantitative PCR were used to detect the efficience of the three siRNA.At last,the efficiency siRNA fragment of integrin-αv gene was selected.