Gelgreen stain

Amplification of the hypervariable 3 region of the 16S rRNA gene from the purified nucleic acids preparations was carried out by PCR reactions and thermal programs where performed as previously described (Davila-Vazquez et al. 2011 (link)), the PCR primers used were the forward primer C356F (5′-CTACGGGAGGCAGCAG-3′) and the reverse primer 517R (5′-ATTACCGCGGCTGCTGG-3′). The primer C356F contains the GC clamp led to carry out the DGGE. The PCR product was loaded onto a 1.5% (w/v) agarose gel with 100 bp molecular weight marker and stained with GelGreen™ stain (Biotium, California, USA) to assess the size (236 bp), purity and concentration of DNA.

Genomic DNA was obtained from a sample of peripheral blood collected in EDTA. The Biopur Mini Spin kit (Biometrix) was used for the DNA extraction. The polymorphisms were determined using the Cytokine Genotyping Tray kit (One Lambda) which employs polymerase chain reaction-sequence specific primers (PCR-SSP), followed by electrophoresis in 2.5% agarose gel stained with GelGreen Stain (Biotium). We investigated the polymorphism TNF-α  -308G/A, IL-10 -1082G/A, IL-10 -819T/C, IL-10 -592C/A, TGF-β1 codon 10T/C, TGF-β1 codon 25C/G, IL-6 -174G/C, and IFN-γ +874T/A, which were selected due to evidence from previous studies that showed their association with the regulation in cytokines gene expression.
For association analyses of polymorphisms, patients were divided into groups according to the complication (DRP, DNP, and DNR) and comorbidity presented (hypertension, dyslipidemia, and BMI—categorized into BMI < 25 Kg/m², BMI 25–30 Kg/m², and BMI ≥ 30 Kg/m²).