Rn00562126 m1

All WAT samples were processed in fresh, 100 mg was disrupted using the TissueLyser LT (Qiagen, Germantown, MD, USA), and total RNA was isolated with the RNeasy^® Lipid Tissue Mini Kit, according to manufacturer’s instructions (Qiagen). The RNA integrity was evaluated on agarose 1% gels stained with ethidium bromide. In addition, the purity and concentration was measured by spectrophotometry.
Total RNA was reverse-transcribed using the RevertAid H minus first strand cDNA Kit (ThermoFisher Scientific, Waltham, MA, USA) as recommended by the manufacturer. cDNA was then amplified using validated TaqMan assays from Applied Biosystems (UCP1: Rn00562126_m1, uncoupling protein 2 (UCP2): Rn01754856_m1 and uncoupling protein 3 (UCP3): Rn00565874_m1) (Applied Biosystems, Foster City, CA, USA). GAPDH (Rn01775763_g1) and Rn45s (Rn03928990_g1) (Applied Biosystems) were used as house-keeping gene. qRT-PCR was performed for each target and house-keeping genes on a StepOne Plus thermocycler (Applied Biosystems). Triplicate cycle threshold C_t values were analyzed using the comparative C_t method (ΔΔC_t) and then presented as relative quantification to GAPDH mRNA expression (RQ) units.

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.

Total RNA was extracted from the hypothalamus, pituitary, and interscapular BAT according to the Tri-Reagent protocol [50 (link)]. The reverse transcription reaction was carried out on 2 μg of RNA using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using TaqMan PCR Master Mix and the thermocycler parameters recommended by the manufacturer. PCRs were performed in a volume of 50 μL, containing 25 μL of the reverse transcription reagents. TaqMan gene expression assays were used for ACCα, CPT1b, FASN, GH, GLUT4, IGF-IR, PPARγ, SRIF, sst2, and UCP-1 (Rn00573474_m1, Rn00682395_m1, Rn00569117_m1, Rn01495894_g1, Rn00562597_m1, Rn01477918_m1, Rn00440945_m1, Rn00561967_m1, Rn00571116_m1 and Rn00562126_m1, respectively; Applied Biosystems). Relative gene expression comparisons were carried out using an invariant endogenous control (actin, Rn00667869_m1). The ΔΔCT method was used for relative quantification.