摘要
通过对兰州大尾羊尾部、大网膜、肝脏、肾周脂肪组织中的总RNA提取,反转录获得总cDNA样品,结合PCR技术,建立了SYBR荧光定量PCR法检测兰州大尾羊心脏型脂肪酸结合蛋白(H-FABP)基因mRNA在不同组织中相对表达量的试验方法,并进行批内、批间重复性检验。结果表明,H-FABP和18S基因标准曲线回归方程分别为CtH-FABP=-3.24375x+38.34230和Ct18S=-3.33137x+33.4573,相关系数(r2)分别为0.9964和0.9992,扩增效率分别为103%和99%;批内、批间重复性测定的变异系数分别为小于1.8%和10%;说明该方法灵敏度高、特异性强、准确可靠、重复性好,是实时荧光定量PCR检测兰州大尾羊心脏型脂肪酸结合蛋白基因mRNA表达量的有效方法。
The method for detecting Lanzhou fat-tailed sheep fatty acid-binding proteins gene mRNA expression with real time PCR was developed.By isolated total RNA form the fresh omentum,kidney weeks adipose tissue,muscle and fat tissue sample using improved the extraction Trizol one step method and reverse transcribed to cDNA using random primers as primer,the results show,the standard curve were CtH-FABP =-3.24375x＋38.34230 and Ct18S=-3.33137x＋33.4573,had good linear dependence,and it was sensitive and specific.The coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged less than 1.8% and 10% respectively.The method for detecting Lanzhou fat-tailed sheep H-FABP gene expression with fluorescence quantitative polymerase chain reaction was developed successfully.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第6期84-88,共5页
China Animal Husbandry & Veterinary Medicine
基金
甘肃省农业生物技术研究与应用开发项目(GNSW-2009-13)
西北民族大学研究生科研创新项目(ycx09061)
