Liver RNA was extracted using the RNeasy Plus Universal Mini Kit (Qiagen, Cat. 73404). Reverse transcription was performed using the Maxima cDNA Synthesis Kit (Thermo Fisher Scientific, Cat. K1681). Generated cDNA was amplified using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Cat. 172–5271) in a Bio-Rad CFX RT-PCR detection system. The final volume of RT-qPCR reaction mix was 20 µL that contains 25 ng of cDNA (5 µL), 450 nM of the forward and reverse primers (1.8 µL), 1X SYBR Green (10 µL), and water (3.2 µL). We randomly selected WT (n=5) and E4 (n=4) mice as biological replicates for RT-qPCR analysis. Duplicates (n=2 technical replicates) were run for all the samples and average Ct values were calculated. The relative mRNA expression was determined using the 2ˆ(−ΔΔCt) method while the Tbp1 or beta-2 microglobulin (B2m) gene was used as a reference gene. Gene primer list and sequences are as described previously.17 (link)