Lightcycler 1536

Total RNA was extracted from RET WAT with Rneasy lipid tissue mini kit from Qiagen. RNA was quantified by spectrophotometric Nanodrop. Total RNA (500ng) was reverse transcribed using SuperScript® III First-Strand Synthesis SuperMix kit from Life Technologies. Samples of cDNA were diluted 1:40, amplified and used for RT-qPCR measurements using SYBR Green from Roche as previously described.¹⁴ (link) RT–qPCR was performed in the LightCycler1536 instrument from Roche as follows: 75°C for 2min, 95°C for 10min, 40 cycles of denaturation (95°C for 15s to the denaturation, 60°C for 30s to the annealing, 72°C for 30s to the extension). Results were analyzed with the LightCycler1536 software from Roche. Ribosomal RPL13 RNA was used to normalize cDNA. Quantification of mRNA was carried out by comparing the number of cycles required to reach reference and target threshold values (2^-(δ−δ Ct) method) as described previously.⁴³ (link) Sequences of the rat sense and antisense nucleotides were: PGC1-α : 5′-tgcccctgccagtcacagga-3′ 5′-gctcagccgaggacacgagg-3′; PPARα : 5′-aagccatcttcacgatgctg-3′ 5′-tcagaggtccctgaacagtg-3′ ; PPARγ2 : 5′- ttatgctgttatgggtgaaa-3′ 5′- caaaggaatgggagtgtc-3′ ; TFAM : 5′-gttccggggaatgtggggcg-3′ 5′-gacaggcgagggtatgcggc-3′ ; UCP1 : 5′-tacagagttatagccaccaca-3′ 5′-tggaacgtcatcatgtttgtg-3′ ; RPL13 : 5′-tggcaggggcttcag-3′ 5′-tgggcatcacaggtcc-3′