Quantitative Real-Time PCR Analysis

After 30 minutes, 1 hour or 6 hours of stimulation by the indicated peptide/hormone (10⁻⁷ or 10⁻⁹ M of ANP, 10⁻⁷ M of isoproterenol, or 0.5 µM of CL316,243), each dish was snap frozen. Where indicated, 20 µM of SB203580 or equivalent volume of DMSO was added to the medium. 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^{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 UCP1 (Applied Biosystems, Rn00562126_m1) and GAPDH (Applied Biosystems, Rn01775763_g1). The transcriptional levels were determined using the ∆∆Ct method with normalization to GAPDH.