Isolation and Gene Expression Analysis of Human Adipocytes

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).

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Santiago-Fernández C., Martín-Reyes F., Tome M., Gutierrez-Repiso C., Fernandez-Garcia D., Ocaña-Wilhelmi L., Rivas-Becerra J., Tatzber F., Pursch E., Tinahones F.J., García-Fuentes E, & Garrido-Sánchez L. (2021). Oxidized LDL Increase the Proinflammatory Profile of Human Visceral Adipocytes Produced by Hypoxia. Biomedicines, 9(11), 1715.

Publication 2021

RNeasy Lipid Tissue Mini Kit Applied Biosystems 7500 Fast Real-Time polymerase chain reaction System SDS software 2.3 RQ Manager 1.2

Adipocytes Assays Cyclophilin a Frozen Gene expression Genes Glut1 Human Lipid Real time polymerase chain reaction Tissue Tnfα

Corresponding Organization : Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas

Other organizations : Hospital Regional Universitario de Málaga, Hospital Clínico Universitario Virgen de la Victoria, Medical University of Graz, University of Applied Sciences Technikum Wien

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Variable analysis

independent variables

Tissue type (frozen human mature adipocytes and in vitro-differentiated adipocytes)

dependent variables

Gene expression of MSR1, CXCL16, LOX-1, CL-P1, HIF-1α, GLUT1, IL6, and TNFα

control variables

Expression of cyclophilin A (PPIA) for normalization

controls

No explicit mention of positive or negative controls

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