4.6.3. Relative quantification of gene expression by real-time PCR

Whole hippocampal tissue was placed in TRIzol reagent according to manufacturer's instruction (Life Technologies) and stored at −80 °C until RNA extraction. Total RNA was extracted using a Qiagen Lipid Tissue Mini Kit Cat. No. 74804, and aliquots per gene were prepared for real time RT-PCR confirmation of those data. RNA quality and quantity were measured by Nanodrop Spectrophotometer ND-1000 UV/Vis. Real-time RT-PCR (n = 7–8) was used to confirm the mRNA levels of differentially expressed genes explained by RNA sequencing in the hippocampus (Abc1, Hmgcr, Cyp46al, Cyp27a1, Srebf1, Srebf2, ApoE, LxrB, Lrp1, and Ldlr). All 10 genes were run on at least 3 replicates across 2 PCR runs, totaling 6 runs/gene/sample. Reverse transcription of RNA was performed by using primers generated by PrimerQuest Tool (Integrated DNA Technologies). Total RNA extraction was performed as aforementioned. cDNA synthesis was performed using a Bio-Rad iScript cDNA synthesis kit (Catalog # 1708891) as previously reported (Sriramula et al., 2008). Gene expression was calculated by ΔΔCT and was normalized to GAPDH mRNA levels. These data are presented as fold change of the gene of interest relative to control animals. Table of primers (Supplemental Table 1).