Quantitative PCR Analysis of Gene Expression

Total RNA from endothelial cells or aorta tissue from mice were isolated using Trizol reagent according to the manufacturer’s protocol. cDNA was synthesized using a High Capacity cDNA reverse transcription kit [28 (link),51 (link)]. The expressions of IL-1 beta, IL-6, TNF-alfa and MCP-1 in endothelial cells and the expression of endothelial nitric oxide synthase (Nos3), inducible nitric oxide synthase (Nos2), Nrf-2 factor (Nfe2l2), glutathione peroxidase-1 (Gpx1) and superoxide dismutase-2 Mn (Sod2-Mn) in aortas from mice were determined by quantitative PCR (ABI Prism 7500 Fast Real-Time PCR System) and analyzed with 7500 Fast sequence detection software v1.3.1 (Applied Biosystems Inc., Foster City, CA, USA), using specific TaqMan assays and Double delta Ct method. TaqMan probes used for mice were Nos3 (Mm00435217_m1), Nos2 (Mm00440502_m1), Nfe2l2 (Mm00477784_m1), Gpx1 (Mm00656767_g1), Sod2-Mn (Mm01313000_m1) and Actb (Mm01205647_g1), and for human cells they were IL1B (Hs01555410_m1), IL6 (Hs00174131_m1), CCL2 (Hs00234140_m1), TNF-alfa (Hs00174128_m1) and ACTB (Hs99999903_m1).

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Asenjo-Bueno A., Alcalde-Estévez E., El Assar M., Olmos G., Plaza P., Sosa P., Martínez-Miguel P., Ruiz-Torres M.P, & López-Ongil S. (2021). Hyperphosphatemia-Induced Oxidant/Antioxidant Imbalance Impairs Vascular Relaxation and Induces Inflammation and Fibrosis in Old Mice. Antioxidants, 10(8), 1308.

Publication 2021

Prism 7500 Fast Real-Time PCR System 7500 Fast sequence detection software v1.3.1

Aortas Assays Ccl2 Cdna Cells Endothelial cells Endothelial nitric oxide synthase Glutathione peroxidase 1 Human Il 1 beta Il1b Inducible nitric oxide synthase Mice Nfe2l2 Nitric oxide synthase Nos3 Nrf 2 Prism Reverse transcription Superoxide dismutase 2 Tissue Tnf alfa Trizol

Corresponding Organization : Fundación Renal

Other organizations : Universidad de Alcalá, Hospital Universitario de Getafe

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Variable analysis

independent variables

Cell type (endothelial cells or aorta tissue from mice)

dependent variables

Expression levels of IL-1 beta, IL-6, TNF-alfa, and MCP-1 in endothelial cells
Expression levels of Nos3, Nos2, Nrf-2 factor (Nfe2l2), Gpx1, and Sod2-Mn in aortas from mice

control variables

Total RNA isolation using Trizol reagent
CDNA synthesis using High Capacity cDNA reverse transcription kit
Quantitative PCR analysis using ABI Prism 7500 Fast Real-Time PCR System and 7500 Fast sequence detection software v1.3.1
Endogenous control genes (Actb for mice, ACTB for human cells)

positive controls

None specified

negative controls

None specified

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