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).