不同荧光定量PCR分析方法检测大鼠脑缺血再灌注损伤中Caspase-3基因的表达研究

目的:应用实时荧光PCR检测大鼠脑缺血/再灌注损伤组织中的Caspase-3基因表达变化,并比较了相对定量与绝对定量分析方法的技术优劣.方法:相对定量实验中,以β-actin基因作为内参,采用Delta-delta Ct法计算目的基因表达变化.而在绝对定量实验中,构建了含Caspase-3基因的重组质粒,以该重组质粒作...

Studies on Expression of Caspase-3 Gene in Rat Cerebral Ischemia-Reperfusion Injury by Two Different Quantification Methods of Real-time PCR

Objective：Relative and absolute quantification methods of real-time PCR were used to investigate the expression of Caspase-3 in rat after Cerebral isocheimal-reperfusion injury. Methods ： In relative quantification experiment, Delta-delta Ct method was adopted by β-actin as a reference gene. In absolute quantification experiment, a recombinant plasmid which includes the Caspase-3 gene was constructed and as a standard to establish the standard curve. The copy number of target gene was calculated by standard curve. Result： Data of relative quantification showed that the Caspase 3 gene was respectively up-regulated 7.52,4.20,3.07 folds in MACO model, treatment group 1 and 2 comparing to sham group. Data in absolute quantification showed that copy number of Caspase-3 gene in sham group was 1. 42 × 10^5 copies/p,L, however in MACO model group it increased to 8.06 × 10^5 copies μL, up-regulated by 5.68 folds. In treatment group 1 and 2, copy number of Caspase-3 gene increased to 3.83 × 10^5 copiesμL and 3.59 × 10^5 copies/μL, 2.71 and 2.52 fold increase respectively comparing to the sham group. Conclusion：The above results indicated that two quantification methods of real-time PCR had compatibility. Absolute quantification method was more accurate than relative quantification, but it needs standard DNA and high cost. Relative quantification method was simple, but its result can＇ t be reliable unless the efficient of target gene was identical to reference gene.