大鼠血管组织血管紧张素原基因表达的实时PCR定量分析

QUANTIFICATION OF mRNA EXPRESSION FOR ANGIOTENSINOGEN IN RAT ARTERIAL TISSUES BY REAL-TIME PCR

目的:探讨大鼠血管组织血管紧张素原(angiotensinogen,AGT)低丰度mRNA表达的实时PCR定量分析方法,并将其用于检测模拟失重大鼠基底和股动脉血管组织AGTmRNA的表达。方法:提取8周模拟失重(SUS)与对照组(CON)大鼠血管组织的总RNA,进行反转录后,对目的基因AGT与内参照基因GAPDH的mRNA进行实时PCR分析。应用TaqMan MGB探针,测出上述mRNA实时PCR反应的放大效率(E)及阈循环数Ct,再依据一定数学模型由E与Ct得出经GAPDH归一化的AGTmRNA表达变化。结果:与CON相比,SUS大鼠基底动脉组织AGTmRNA表达增加240%,而股动脉组织则降低66%。结论:本工作为定量检测大鼠血管组织低丰度mRNA表达提示了一种特异、灵敏、精确、重复性好的简便方法。

Aim: To develop a real-time PCR method for quantifying low abundance mRNA expression in rat arterial tissues and examine differential changes of angiotensinogen(AGT) gene expression in basilar and femoral arterial tissues after 8weeks simulated weightlessness. Methods: After 8-wk of simulation, basilar and femoral arteries were harvested from both simulated weightless(SUS) and control(CON) rats. Then total RNA was extracted and reverse transcribed. The real-time PCR using TaqMan-MGB probe was performed to quantify AGT mRNA expression. The amplification efficiency(E) of PCR was calculated from the slope of standard curve. The threshold cycle(Ct) was detected by changes in fluorescence during a real-time PCR. Finally, the relative expression ratio of the target gene(AGT) to the reference gene(GAPDH) was calculated using E and Ct according to the mathematical model derived from the equation calculating the starting fluorescence(Ro). Results: After a 8-weeks simulated weightlessness, the AGT mRNA expression increased by 240 percent in basilar arterial, and decreased by 66 percent in femoral arterial tissues. Conclusion: The specificity, sensitivity, precision, reproducibility, and simplicity of real-time PCR method using TaqMan-MGB probe make it particularly suitable for quantification of low abundance mRNA in arterial tissue from rats.