Quantitative reverse transcription polymerase chain reaction

Total RNA from liver tissue samples of 24 male and female CC mouse strains [²²] was isolated using miRNeasy Mini kits, and 2 μg of total RNA was reverse transcribed using random primers and High-Capacity cDNA Reverse Transcription kits (Life Technologies, Grand Island, NY). The expression of selected differentially methylated genes was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the TaqMan gene expression assays that are listed in Supplementary Table 2. The glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene was used as an endogenous control. The relative amount of each mRNA transcript was determined by the 2^−ΔΔCt method [²⁹]. A p-value cut-off of <0.05 and a fold change threshold of >1.5 were used to generate a list of DEGs in the livers of mice fed the HF/HS diet.