Sensifast sybr green mix

RNA was extracted from frozen liver tissue using ice-cold TRIzol reagent (Life Technologies) according to the manufacturer's protocol, followed by DNase I treatment (Roche). Complementary DNA was synthesized by standard reverse transcription (High Capacity cDNA synthesis kit, Roche) on a MultiGene thermal cycler (Labnet). Amplification and semi-quantification of transcripts were performed using Sensifast SYBR Green mix (Bioline) on a real-time PCR system (iCycler, Bio-Rad) with mouse gene-specific primers (Integrated DNA Technologies). Cyclophilin A (CypA) was used as a housekeeping gene. Primers used were CypA (F: 5′-CGATGACGAGCCCTTGG-3′, R: 5′-TCTGCTGTCTTTGGAACTTTGTC-3′), alpha-fetoprotein (Afp; F: 5′-CCCGCTTCCCTCATCC-3′, R: 5′-GAAGCTATCCCAAACTCATTTTCG-3′), glucose-6-phosphate dehydrogenase (G6pd; F: 5′-AAGAAGCCTGGCATGTTCTT-3′, R: 5′-GAAGCCCACTCTCTTCATCA-3′), lipoprotein lipase (Lpl; F: 5′-GGATGGACGGTAACGGGAAT-3′, R: 5′-ATAATGTTGCTGGGCCCGAT-3′), Cd36 (F: 5′-GATGACGTGGCAAAGAACAG-3′, R: 5′-TCCTCGGGGTCCTGAGTTAT-3′), fatty acid transport protein 5 (Fatp5; F: 5′-GCACCTTCTGACCCAGTACC-3′, R: 5′-GTAAGCAGCCAAGGAATCCA-3′).

Measurement of gene expression by qRT-PCR was performed as previously outlined^{43 (link)}. Briefly, total RNA was isolated using Trizol or Direct-zol (Zymo Research) and used for cDNA synthesis (2 μg liver RNA or 1–2 μg cell RNA) by two-step RT-PCR with the High Capacity cDNA synthesis kit (Roche). Levels of mRNA were semi-quantified with Sensifast SYBR Green mix (Bioline) and gene-specific primers (Integrated DNA Technologies) and were normalized to the housekeeping gene Ppia (CypA). For specific primer sequences, see Supplementary Table 1.

Real-time quantitative RT-PCR (RT-qPCR) was performed to evaluate RNA expression. Total RNA was extracted using Nucleospin RNA isolation kits (Macherey-Nagel, Germany), according to the manufacturer’s protocol, and reverse-transcribed using Moloney murine leukemia virus reverse transcriptase with random hexamer primers or oligo(dT) primer (Promega, Madison, WI). RT-qPCR analysis was performed using the SensiFast Sybr green mix (Bioline, London, UK) on a LightCycler system (LC480, Roche Applied Science, Penzberg, Germany). LinRegPCR software was used to analyze the data (Untergasser et al., 2021 (link)). Primers used are shown in Supplementary Table 1.