Nanodrope

The total RNA was extracted from ~ 1 cm² of adipose tissue using QIAzol lysis reagent (Qiagen, Hilden, Germany). The amount of RNA was determined using NanoDrope (Thermo Scientific, Waltham, MA, US) spectroscopy, and diluted to a final concentration of 50 ηg/μl in 20 μl. In total, 1 μl of RNA was used for the synthesis and amplification of complementary DNA (cDNA), which followed the protocol of the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, US). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using the PPAR gamma gene (Mm00440940_m1), and the endogenous control GAPDH (Mm99999915_g1) followed the TaqMan Universal PCR Master Mix Kit (Applied Biosystems) using the StepOne Real-Time PCR System (Life Technologies, Foster City, CA, US). Real-time PCR reactions were conducted as follows: after a pre-denaturation and polymerase-activation program (2 minutes at 50°C and 10 minutes at 95°C), 50 cycles, each one consisting of 95°C for 15 seconds and of 60°C for 1 minute. The negative controls consisted of wells in which the cDNA was absent. The relative expression of PPAR gamma/GAPDH was calculated using the equation ΔCt, which expresses the difference between the number of threshold cycles (Cts) of the target genes and the endogenous control.

The feces were kept frozen at −80°C until the extraction process. DNA extraction assay was performed according to the Fecal DNA extraction International Human Microbiome Standards (IHMS) Protocol H (http://www.microbiome-standards.org/). However, an adaptation of this protocol was made before step 16: Chloroform (1:1) was added in supernatants, mixed for 5 min, and then centrifuged at 12,000 rpm for 10 min. The supernatant was collected without intermediates layer contaminants. After this step, all the next procedures were performed exactly as described in the IHMS protocol. The DNA was quantified using the Qubit 3.0 fluorometer (Thermofisher Scientific). The protein contamination was evaluated by the A260/A280 ratio using nanodrope (Thermofisher Scientific), and DNA integrity was evaluated by 0.6% agarose gel electrophoresis.