Human visceral adipose tissue RT-PCR in individuals with obesity

Visceral adiposity is more related with insulin resistance than BMI itself. To study the molecular signature of the subsequent evolution steps of the metabolic sequalae of obesity we collected omental adipose tissue samples from individuals with obesity while undergoing bariatric surgery. Participants (all with obesity) were divided in 4 groups according to glycemic profile: Insulin sensitive/normoglycemic group (IS NG) - OxHOMA2<1 and fasting glycemia < 100 mg/dL (n=17); Insulin resistant/normoglycemic group (IR NG) - OxHOMA2 > 1 and fasting glycemia < 100 mg/dL and HbA1c < 5.7% (n=24); Pre diabetes group (PD) - OxHOMA2 > 1 and fasting glycemia > 100 mg/dL or HbA1c [5.7% - 6.4%] (n=33); Type 2 diabetes group (T2D) - OxHOMA2 > 1 and fasting glycemia > 125 mg/dL or HbA1c > 6.4% (n=18). A panel of 43 genes related to inflammation, angiogenesis, lipid metabolism and redox machinery was evaluated in a BioMark HD Fluidigm system. Samples of visceral adipose tissue were collected from the greater omentum while undergoing metabolic surgery, which was always performed by the same appointed surgeon. Adipose tissue biopsies were collected from the patients and kept in liquid nitrogen during transportation to our facilities, to be stored at -80 ºC. Total RNA was isolated from human visceral adipose tissue biopsies (100 mg of tissue) with a RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Upon isolation, RNA samples were analyzed by NanoDrop One/One spectrophotometer (ThermoFisher, Waltham, MA, USA) at 260nm to evaluate RNA concentration. Each RNA sample was diluted to the same concentration (25 ng/μL). 1μl of Reverse transcriptase enzyme [qScript cDNA super mix (Quanta BioSciences)] was added to 4μl of each RNA sample to obtain cDNA. Each cDNA sample was pre-amplified (PA). Briefly, 1.25μl of each cDNA sample was mixed with 0.5μl of a mix of pooled primers (500nM final concentration each), 2.25μl water and 1μl of PreAmp Master Mix enzyme (Fluidigm). Thermal cycling was performed according to the enzyme manufacturer for 12 cycles. The samples were then treated with Exonuclease I (New England Biolabs) to remove unincorporated primers. After Exonuclease treatment the samples were diluted 5x in TE buffer (10mM Tris-HCl, 1mM EDTA). For each sample (2.25μl) a Pre-Mix was prepared with 2.5μl SsoFast Eva Green Supermix (BioRad), 0,25μl of 20x DNA binding dye sample reagent (Fluidigm). 5μl of each sample was pipetted into the respective inlet of a Fluidigm® 96.96 Gene expression integrated fluidic circuit (IFC). For each assay (gene) a mix of 12μl was individually prepared: 6μl of 2x Assay loading reagent (Fluidigm), 5.4μl of TE buffer, 1.2 μl from a stock of 50μM each mixed forward and reverse primers. 5μl of each assay was pipetted into their respective assay inlets on the chip. The assay and sample mixes were loaded with the Load mix (136x) script of the HX controller (HD Biomark). After loading, the chip qRT-PCR was carried out using the BioMark HDTM, accordingly to the cycling parameters recommended by Fluidigm® for 96.96 Gene expression IFC. Data were collected with Data Collection Software and were analyzed using Fluidigm® Real Time PCR Analysis v2.1 software. Genes with melting curves displaying more of one peak (amplification of non-specific products) were not included in the analysis. The data were normalized for the reference gene ATCB and ddCt was calculated.The oligos were designed for human transcripts and were synthesized by Sigma-Aldrich and reconstituted to a final concentration of 100 µM in water.