Quantitative RT-PCR Analysis of Cardiovascular Genes

Total mRNA from aortic and brain tissue was isolated using the acid guanidinium thiocyanate–phenol–chloroform extraction method. 125 ng of total RNA was used for quantitative reverse transcription real-time PCR (qRT-PCR) analysis using QuantiTect Probe RT-PCR kit (Qiagen) as described previously [22 (link), 51 (link)]. Primer–probe–mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase 2 (NOX-2 Mm00432775_m1), eNOS (NOS3, Mm00435204_m1) and vascular cell adhesion protein 1 (VCAM-1, Mm00449197_m1) in aorta and NFκB2 (NFκB2, Mm00479810_g1), CD40L (CD40L, Mm_00441911_m1) in brain. All samples and tissues were normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control. For quantification of the relative mRNA expression, the comparative ΔΔCt method was used. Expression of target gene in each sample was expressed as the percentage of unexposed wild type.