RNA Extraction and qPCR Analysis in Adipose and Brain Tissue

RNA was extracted from visceral adipose tissue or the cerebral cortex by lysis with TRIzol reagent (Life Technologies, Carlsbad, CA, USA, Cat #15596018), according to the manufacturer’s protocol, except for in adipose tissue. For adipose tissue, tissue was lysed using a dounce homogenizer for 20 strokes. The solution was centrifuged for 10 min, and the top layer (fat) was removed prior to the addition of chloroform. After the completion of the TRI-zol protocol, potential genomic DNA contamination was removed from all samples by an RNase-free DNase treatment (Lucigen, Middlesex, UK, Cat #D9905K). Purified RNA (1 μg) was used for reverse transcription using the iScript synthesis system (Bio-Rad, Hercules, CA, USA, Cat #1708891). The resulting cDNA was used for quantitative PCR using a Bio-Rad CFX96 Touch Real-Time PCR Detection System. Standard PCR cycling protocols were used, consisting of 40 cycles, with denaturation at 95 °C and annealing at 60 °C. The amplification efficiency was estimated from the standard curve for each gene. All primers have an efficiency of 88–115%. Relative quantification of mRNA levels was determined by the ΔΔCt method. All groups were compared to the APOE3 control. All experimental primers were compared to the expression of β-actin in the brain and the average expression of succinate dehydrogenase complex, subunit A (SDHA), and hypoxanthine guanine phosphoribosyltransferase (HPRT) in adipose tissue. The expression of control primers showed no significant differences according to the APOE genotype or diet (2-way ANOVA). Primer sequences (Life Technologies) are listed in Table 1.