AIMTo 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 8 weeks simulated weightlessness.

METHODSAfter 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).

RESULTSAfter 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.

CONCLUSIONThe 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.