RNA extraction, amplification and labelling from subcutaneous adipose tissue biopsies

Total RNA was isolated from adipose tissue using the RiboPure kit (Applied Biosystems) after homogenizing the RNALater-stabilized tissues in TRI Reagent. The quantity and quality of the RNA samples were determined using a NanoDrop ND-1000 spectrophotometer. Samples whose A260/A280 ratio deviate ±0.2 from the accepted ratio of 2.0 were excluded. The quality/integrity of the RNA was assessed using the Agilent RNA 6000 Nano LabChip Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies), ensuring that the 28 S and 18 s ribosomal RNA species are intact and that significant degradation had not occurred. The concentration of the resulting RNA samples was determined by using the NanaoDrop ND-1000 spectrophotometer. For synthesis of cRNA, the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) was used. The quality of the cRNA was assessed using the Agilent RNA 6000 Nano LabChip Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies). cRNA quantity was measured using a NanoDrop ND-1000 spectrophotometer.