兰州大尾羊解偶联蛋白3(UCP3)基因荧光定量PCR检测方法的研究

Establishment of the method for detecting Lanzhou fat-tailed sheep uncoupling protein 3 (UCP3) gene mRNA with fluorescence quantitative ploymerase chain reaction

试验通过提取兰州大尾羊不同组织中总RNA,反转录获得总cDNA样品,建立了用SYBR荧光定量PCR法检测兰州大尾羊解偶联蛋白(UCP3)基因mRNA在不同组织中的相对表达量的检测方法,并进行批内、批间重复性检验。结果表明:UCP3和GADPH基因标准曲线回归方程分别为CtUCP3=-3.2343x+41.6198和CtGADPH=-3.2851x+38.0344,相关系数(r2)分别为0.9958和0.9986,扩增效率分别为103%和97%;批内、批间重复性测定的变异系数分别为小于1.4%和10%。

The experiment was conducted to the method for detecting Lanzhou fat-tailed sheep uncoupling protein 3 （UCP3） gene mRNA expression with real-time PCR developed.By isolated total RNA form the fresh omentum,kidney weeks adipose tissue,muscle and fat tissue,the sample was improved the extraction Trizol one-step method and reverse-transcribed to cDNA using random primers as a primer.The results demonstrated that the standard curve were CtUCP3=-3.234 3x＋41.619 8 and CtGADPH =-3.285 1x＋38.034 4,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 1.4% and 10% respectively.