2.5. RNA Isolation and qPCR of Nfe2l2 and ER Stress Target Genes

Total RNA was isolated from livers of WT and NRF2 KO mice fed either Con or MR diets using RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA concentrations were measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA was analyzed using the Agilent Bioanalyzer RNA 1000 chip as a quality control step to confirm the integrity of the RNA. Samples were verified to have RIN values > 7, indicating high quality RNA. Two μg of total RNA was used for reverse transcription to produce cDNA. mRNA expression for previously identified Nfe2l2- and ER stress-target genes [25] was measured using 10 ng cDNA via quantitative PCR using SYBR Green (Bio-Rad, Valencia, CA, USA), and mRNA concentrations of each target gene were standardized to cyclophilin expression. Primer sequences are provided in Supplementary Table S1.