Invitrogen rnaseout recombinant ribonuclease inhibitor

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.

Total RNA was reverse transcribed using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific) and Invitrogen™ RNaseOUT™ Recombinant Ribonuclease Inhibitor (Fisher Scientific) following the instructions as recommended by the manufacturer to synthesise cDNA, performed using the T100™ Thermal cycler (Bio-Rad). The gene expression of 30 ng cDNA was determined using 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 fold change in gene expression levels were determined using the comparative threshold cycle (CT) method, also referred to as the 2^−ΔΔCt method. The target genes listed below were normalized to the housekeeping gene, peptidylprolyl isomerase A (PPIA). All Applied Biosystems™ TaqMan™ Gene Expression Assay (FAM-MGB) (Table 1) were purchased from Fisher Scientific.