Applied biosystems 7500 fast real time polymerase chain reaction system

The expression by RT-qPCR of 6 miRNAs in the same 8 samples (n = 4 per group) (technical validation) or in 16 samples (the same 4 samples per group plus another 4 samples per group) (biological validation) were analyzed on the basis of their ratio ranking and their biological interest. It was using the TaqMan^® MicroRNA Reverse Transcription kit following the manufacturer's instructions (Thermo Fisher Scientific Inc., Waltham, MA). miRNA expression was assessed by real-time PCR using an Applied Biosystems 7500 Fast Real-Time polymerase chain reaction System (Applied Biosystems, Foster City, CA). Reactions were carried out in duplicate for all miRNAs using TaqMan™ MicroRNA Assay (Thermo Fisher Scientific Inc., Waltham, MA): hsa-miR-1-3p (477820_mir), hsa-miR-216a-5p (477976_mir), hsa-miR-31-3p (478012_mir), hsa-miR-20a-5p (478586_mir), hsa-miR-126-5p (477888_mir), and hsa-miR-204-5p (478491_mir). The threshold cycle value for each sample was normalized with the expression of hsa-miR-1-3p (Ref: 477820_miR). We chose this miRNA because it was the endogenous control tested with a more constant expression (with the lowest coefficient of variation) and with greater expression. SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems, Foster City, CA) were used to analyse the results with the comparative Ct method (2^−ΔCt). The Log₂ FC of EVOO vs. SO were determined.

Total RNA from frozen human mature adipocytes and in vitro-differentiated adipocytes were isolated using an RNeasy Lipid Tissue Mini Kit (Qiagen, GmbH, Hilden, Germany) as previously described [23 (link),27 (link),29 (link)]. Gene expression was assessed by real-time PCR using an Applied Biosystems 7500 Fast Real-Time polymerase chain reaction System (Applied Biosystems, Darmstadt, Germany). The reactions were carried out in duplicate for all genes using specific TaqMan^® Gene Expression Assays: MSR1 (Hs00234007_m1, RefSeq. NM_002445.3, NM_138715.2, NM_138716.2), CXCL16 (Hs00222859_m1, RefSeq. NM_001100812.1, NM_022059.2), LOX-1 (Hs01552593_m1, RefSeq. NM_001172632.1, NM_001172633.1, NM_002543.3), CL-P1 (Hs00560477_m1, RefSeq. NM_130386.2), HIF-1α (Hs00153153_m1, RefSeq. NM_001243084.1, NM_001530.3, NM_181054.2), GLUT1 (Hs00892681_m1, RefSeq. NM_006516.2), IL6 (Hs00174131_m1; RefSeq: NM_000600.3) and TNFα (Hs00174128_m1; RefSeq: NM_000594.3). The threshold cycle (Ct) value for each sample was normalized with the expression of cyclophilin A (PPIA) (4326316 E, RefSeq. NM_021130.3) [1 (link)]. SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems, Foster City, CA, USA) were used to analyze the results with the comparative Ct method (2^−ΔCt).