*3.6. RNA Extraction and Quantitative Real-Time RT-PCR*

Total RNA of eWAT, rWAT, iWAT and BAT were isolated by an RNeasy® Plus Universal Mini Kit (*Qiagen*, Stockach, Germany), according to the manufacturer's instruction. Total RNA (2 ΐg) was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). PCR was performed in a final volume of 25 ΐL containing 10 ΐL cDNA (1 ng/ΐL), 12.5 ΐL TaqMan® Gene Expression Master Mix, 1.25 ΐL probe/primer assay mix and 1.25 ΐL water. PCR primers and probes were purchased from Applied Biosystems: UCP1, CIDEA (cell death-inducing DFFA-like effecter a), Prdm16 (PR domain containing 16), PGC-1΅, NRF1, NRF2, TFAM, ERR΅, PPAR΅, PPAR·, HSL (hormone-sensitive lipase), Ά3-AR (Ά3-adrenergic receptor), Dio2 (deiodinase 2), Mfn1, Mfn2, OPA1 (optic atrophy 1), Fis1 (fission 1) and Ά-actin. These genes are associated with thermogenesis, mitochondrial biogenesis, browning of WAT and mitochondrial fusion and fission. The mRNA expression levels were determined by quantitative 

real-time RT-PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression levels were normalized to Άactin. Data were analyzed by StepOne Software (v2.2.2, Applied Biosystems, Foster City, CA, USA). 
