Gene Expression Analysis

In separated groups of rats, qRT-PCR was performed in the liver. The total RNA from the liver was isolated with TRI reagent^® (Sigma-Aldrich) according to the manufacturer’s protocol. All isolated RNA was quantified by spectrophotometry, and the optical density was estimated from the 260/280 nm absorbance ratio. A reverse transcriptase reaction was performed using SuperScript ™ III (Invitrogen Life Technologies) for first-strand cDNA synthesis. Real-time PCR was carried out following the generation of first-strand cDNA. A PCR for each sample was carried out in triplicate for all cDNAs and for the 18s ribosomal control and were used SYBR^® Green PCR Master Mix (Applied Biosystems, Rockford, USA). The analyzed genes are described in Table 2. The analyses were performed by a relative method for quantifying gene expression (comparative Cq, ΔCq), which allows one to quantify differences among samples in the level of expression of a specific target. The expression levels were normalized for the amount of the reference gene (Rplp2) on each plate. The results were obtained with a formula that considers the amount of the target gene normalized to the calibrator gene, given by (2–ΔCq).

Rat Genome Database (RGD) accession numbers and primer sequences of genes selected for qRT-PCR.

Primers used (Forward and Reverse).