2.4.7. Analysis of gene expression by RT-qPCR
Total RNA from aortic rings, liver, fat and colon samples was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD, USA), following the manufacturer’s recommendations, and was reverse transcribed using oligo(dT) primers (Promega, Southampton, UK). Real time quantitative PCR amplification and detection was performed on optical-grade 48 well plates in EcoTM Real time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) and specific Sigma predesigned primers at their annealing temperature (Table 1). The expression levels of the target genes were normalized to that glyceraldehydes 3-phosphate dehydrogenase (Gapdh ) and measured by the comparative Ct (∆∆Ct).