Quantitative Analysis of Cardiac Gene Expression

After 6 h of stimulation by the indicated hormone (10 nM, 500 nM or 50 μM of T1AM, 100 nM of ET-1, and 20 μM of PE), each dish was snap frozen. Where indicated, NRCM were first pretreated with 500 nM of AZD6244 (Cayman Chemical) or 10 μM of H-89 30 min prior to the indicated hormone stimulation. Total RNA was extracted from the frozen cells using TRIzol reagent (Invitrogen) and a quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as described previously^{15 (link),37 (link)}. The real-time PCR protocol consisted of one cycle at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s using the primers for NPPB (Applied Biosystems, Rn00580641_m1) and GAPDH (Applied Biosystems, Rn01775763_g1). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH^{15 (link)}.