Iq5 program

RNA from cells was extracted in Trizol Ambion (AM9738, ThermoFisher, Madrid, Spain) following the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000 spectrophotometer (ThermoFisher), and 1 µg of RNA was reverse-transcribed to cDNA with a Transcriptor First-Strand cDNA Synthesis kit (04379012001, Roche). qPCR assay was carried out with 5 µL of this template cDNA, 10 µL of the SYBR Green PCR Master Mix cocktail (4309155, ThermoFisher) and 250 nM forward and reverse primers (Supplementary Table S2). RPLP0 (36B4) was chosen as a housekeeping endogenous control for normalization purposes. A qPCR reaction was carried out in MyIQ RealTime PCR System (BioRad, Madrid, Spain). Result analysis was conducted with the IQ5 program (BioRad) following the 2^−ΔΔCt method.

To extract RNA from liver samples, we used PureLink^® RNA Mini Kit, following the manufacturer’s instructions (Ambion^TM, Invitrogen, Carlsbad, CA, USA). Briefly, lysis buffer, supplemented with 1% β-mercaptoethanol, was added to a liver piece and homogenized. The centrifuged supernatant was transferred to a new tube and the same volume of ethanol 70% was added. After mixing, the whole volume was transferred to the mini-columns. The extracted RNA was eluted with RNAse-free water from the column to a proper tube. The samples were stored at −80 °C, prior to conversion to cDNA and qPCR analysis. Total RNA was transcribed into cDNA by using an NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal). For the amplification of each gene of interest, a corresponding specific pair of primers (STAB Vida, Lisbon, Portugal) was used (Table 2). All reactions were performed in a 20 μL total volume with iTaq SYBR green PCR master mix (Bio-Rad, Hercules, CA, USA). Baseline thresholds were obtained by using the Bio-Rad iQ5 program, and the threshold cycles (CTs) were calculated by the 2CT method, where CT values for the genes of interest were normalized to the level of the hypoxanthine-guanine phosphoribosyl transferase housekeeping gene (Hprt1). Data are shown as n-fold differences relative to values for noninfected samples of Fth1^+/+ genotype, calculated with the 2^−ΔΔCT method.

Total cellular RNA was extracted by Tri Reagent (Ambion, Inc.), according to the manufacturer's instructions. RNA quantification was performed using spectrophotometry. One microgram of RNA was used for reverse transcription using GeneAmp RNA PCR Kit (Applied BioSystems) and Random Examers as primers to cDNA synthesis. The primers used for CPT1Av1 and CPT1Av2 amplification reactions are reported in Mazzarelli et al., 2007. All reactions were processed in MyiQ Single-Color Real-Time PCR Detection System (Bio Rad) and results were analyzed by IQ5 program (Bio Rad). To normalize template input, β2-microglobulin (endogenous control) transcript was amplified for each sample.

RNA from cells was extracted in Trizol Ambion (AM9738, Thermo Fisher) following the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000 (ThermoFisher) and 1 µg RNA was reverse-transcribed to cDNA with Transcriptor First-Strand cDNA Synthesis kit (04379012001, Roche). qPCR assay was carried out with 5 µL of this template cDNA, 10 µL SYBR Green PCR Master Mix cocktail (4309155, ThermoFisher) and 250 nM forward and reverse primers (Supplementary Table S2). RPLP0 (36B4) was chosen as a housekeeping endogenous control for normalization purposes. qPCR reaction was carried out in MyIQ RealTime PCR System (BioRad). Result analysis was conducted with the IQ5 program (BioRad) following the 2^-ΔΔCt method.