2.12. Gene Expression Analysis

Total RNA from the liver samples was extracted using TRIzol reagent, and cDNA was generated from 1 µg of total RNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). The sequences of the primers used for the quantitative real-time polymerase chain reaction (qPCR) analysis of targeted genes, including acyl-CoA thioesterase 1 (Acot1), carnitine palmitoyltransferase 1 (Cpt1), phosphatidylethanolamine N-methyltransferase (Pemt), glutamate-cysteine ligase modifier subunit (Gclm), glutamate-cysteine ligase catalytic subunit (Gclc) were designed using the NCBI/Primer-BLAST tool (Supplementary Table S3). The qPCR reactions were conducted using the SYBR green PCR master mix (Thermo Fisher Scientific) in a StepOnePlus system (Applied Biosystems, CA, USA). Using β-actin as the reference gene, the expression levels of genes were quantified using the comparative threshold cycle (CT) method.