RNA extraction and cDNA synthesis

Total RNA in aorta tissue and cells was isolated using RNAiso Plus kits (TAKARA, Tokyo, Japan) [27]. Animal samples and cell samples are mixed with 0.2 ml of chloroform (Amresco, Solon, OH, USA) and then, centrifuged at 13,000 ×g for 15 min at 4°C. Clear part was collected into new tubes and mixed with 0.4 ml of 100% isopropanol and centrifuged at 13,000 ×g for 10 min at 4°C. Isolated RNA pellets were washed with 1 ml of 75% ethanol and centrifuged at 7,500 ×g for 5 min at 4°C. Dried pellets were dissolved in 10 to 50 µl diethyl pyrocarbonate (DEPC, Sigma-Aldrich) treated water and the prepared RNA was quantified using a Nanodrop 2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from RNA using a cDNA synthesis kit (PrimeScript; TAKARA).