Quickprep adaptor

Levels of mRNA were measured exactly as described previously^{38 (link)}. Briefly, interscapular BAT was isolated in Invitrogen TRI Reagent, lysed and homogenized with QuickPrep Adaptor (Fisher Scientific). RNA isolation was performed according to the manufacturer (Fisher Scientific) and cDNA synthesized using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific), Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor (Fisher Scientific) and T100 Thermal cycler (Bio-Rad). Gene expression of cDNA (30 ng) was determined using the qPCRBIO Probe Mix No-ROX (PCR Biosystems), performed on the MyiQ Single-Colour Real-Time PCR Detection System (Bio-Rad). Samples were measured in duplicates and the 2^−∆∆Ct method employed to determine fold change in gene expression. The target genes were normalized to the housekeeping gene, peptidylprolyl isomerase A (PPIA). All Applied Biosystems TaqMan Gene Expression Assay (FAM-MGB) (Major Resources Table) were purchased from Fisher Scientific.

One millilitre (1 mL) of Invitrogen™ TRI Reagent™ Solution per 50 mg to 100 mg of gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), intercapsular brown adipose tissue (iBAT) and pericardial adipose tissue (PAT) was transferred to a MP Biomedicals^TM Lysing Matrix D tube. Lysing tubes were placed in the MP Biomedicals™ FastPrep-24™ 5G Instrument, with the QuickPrep Adaptor (Fisher Scientific) and homogenised at a speed of 6.0 m/s, for 40 s. Isolation of RNA was performed as recommended by the manufactures of Invitrogen™ TRI Reagent™ Solution (Fisher Scientific).