Quantitative gene expression analysis

Total RNA was extracted from BM cells and splenocytes using TRIzol reagent (Invitrogen, MA, USA) and isopropanol precipitation according to manufacturer’s recommendations. cDNA was synthesised from 1.5 μg total RNA using Superscript™ IV reverse transcriptase (Invitrogen) as per manufacturer’s protocol. qPCR was performed on the Quantstudio 3 Real-Time PCR System (Applied Biosystems, MA, USA) using RT² SYBR^® Green reagent (QIAGEN, Hilden, Germany). Gene expression was analysed using the ΔCt method (2^−ΔCt) normalised to RPLP0. RPLP0 normalisation was chosen based on its known stability as a reference gene in HFD-fed C57BL/6J mice^{65 (link)}, and its stability as a housekeeping gene in BM^{66 (link)}. The following primers were used: RPLP0 F: 5′ AGATTCGGGATATGCTGTTGGC 3′, RPLP0 R: 5′ TCGGGTCCTAGACCAGTGTTC 3′, TNFα F: 5′ CCTGTAGCCCACGTCGTAG 3′, TNFα R: 5′ GGGAGTAGACAAGGTACAACCC 3′, IL1β F: 5′ GCCACCTTTTGACAGTGATGAG 3′, IL1β R: 5′ AGCTTCTCCACAGCCACAAT 3′, IL6 F: 5′ TAGTCCTTCCTACCCCAATTTCC 3′, IL6 R: 5′ TTGGTCCTTAGCCACTCCTTC 3′, MPO F: 5′ TCCCACTCAGCAAGGTCTT 3′, MPO R: 5′ TAAGAGCAGGCAAATCCAG 3′, human MYC F: 5′ CGTCCTCGGATTCTCTGCTC 3′, human MYC R: 5′ GCTGCGTAGTTGTGCTGATG 3′.