Random hexamers primers

Total RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen), according to the manufacturer’s protocol. The RNA was treated with RNase-free DNase I (Sigma-Aldrich, St. Louis, MO, USA) to avoid contamination with genomic DNA. Extracted RNA was quantified with Nanodrop™ 2000c spectrophotometer. For template cDNA synthesis, 1 µg of total RNA was reverse transcribed in a 20 µL final volume using random hexamers primers (Roche Applied Science, Penzberg, Germany) and 200 units of M-MLV reverse transcriptase (Invitrogen), as previously described [36 (link)].
Real-time PCR reactions were carried out using a CFX96 Real-Time System C1000 (BioRad, Hercules, CA, USA), the TaqMan Universal PCR Master Mix (Applied Biosystems Thermo Fisher Scientific, Waltham, MA, USA), and the following specific TaqMan pre-designed gene expression primers: AQP1 (Hs01028916_m1), AQP3 (Hs01105469_g1), AQP5 (Hs00387048_m1), and ACTB (Hs99999903_m1) (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). The cDNA was amplified at the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.
The relative quantification of gene expression was determined using the 2^–ΔCt method (adapted from Reference [37 (link)]). Using this method, we obtained the fold variation in AQP gene expression normalized to an endogenous control (β-actin).

We performed total RNA extraction from the whole body or specific tissues if indicated, using the miRNeasy extraction kit (QIAGEN). A 500-ng sample from each RNA extraction was treated with DNase (Promega) and reverse transcribed with first Strand cDNA Synthesis Kit (Roche) and random hexamers primers (Roche). RNA quantity and quality was estimated by spectrophotometric absorption at 260 nm using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies).

Total RNA isolation from VAT was isolated using RNeasy Lipid Tissue Mini Kit according to the manufacturer´s instructions (Qiagen GmbH, Hilden, Germany). To generate first-strand cDNA synthesis, we used 1 μg of total RNA, Moloney Murine Leukemia Virus reverse transcriptase, and random hexamers primers (Roche Diagnostic, Rotkreuz, Switzerland) as indicated by the manufacturer. We used commercially available TaqMan primer/probe mix (Integrated DNA Technologies Inc, Madrid, Spain) for the quantification of C/EBP-α (Hs.PT.58.3605816, RefSeq. NM_001079533), PPAR-γ (Hs.PT.58.4423955, RefSeq. NM_005036), PGC-1α (Hs.PT.58.4461873, RefSeq. NM_002630), NF-κB (Hs00765730_m1, RefSeq. NM_001165412.1 and NM_003998.3), DNTM3a (Hs.PT.58.28037916, RefSeq. NM_001130823), and PPIA (4326316E, RefSeq. NM_021130.3), used as endogenous controls for the target genes. Gene expression was carried out by real-time PCR using an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) with TaqMan technology. Changes of gene expression were calculated by the 2−ΔΔCt method [56 (link)]. The results of the expression were represented as the target gene/PPIA ratio.