Qbaseplus data analysis software

Total RNA from brown adipocytes at differentiation days 1, 3, 6 and 9 was isolated using the RNeasy Mini Kit (Qiagen) before and after incubation with pro-inflammatory cytokines. Isolated RNA was quantified and analysed by NanoDrop 1000 (Thermo Scientific). Two micrograms of RNA were reverse transcribed into cDNA by using random hexamer primers. For quantitative real-time PCR (qRT-PCR), QuantiTect SYBR Green technology (Qiagen) and the ViiA™ 7 Real-Time PCR System (Life Technologies) were used. Samples were incubated at 50°C for 5 min followed by a 10 min denaturation step at 95°C. Following this initial denaturation, the 40 PCR cycles comprised a denaturation step at 95°C for 30 s, an annealing step at 60°C for 30 s and an extension step at 72°C for 30 s. The optimal PCR reaction parameters were defined empirically and the purity and specificity of the amplified PCR product in each experiment was verified by melting curves. Primers were designed to have annealing temperatures of 60°C and to provide amplicons between 79 and 190 base pairs (Supplementary Table S1). All measurements were performed in triplicate and normalized to the housekeeping genes β-actin, peptidylprolyl isomerase A (PPIA) and α-tubulin using the QBasePLUS data analysis software (Biogazelle).