Total DNA and RNA in the caudate and putamen of study brains were extracted separately using the Qiagen QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA; Cat# 51304) and the Qiagen RNeasy Plus Micro Kit (Qiagen; Cat# 74034). The concentrations of 8-oxo-dG and 8-oxo-G were determined separately using the OxiSelect oxidative DNA damage ELISA (Cell Biolabs, Inc., San Diego, CA, USA; Cat# STA-320) and the OxiSelect oxidative RNA damage ELISA kits (Cell Biolabs, Inc., Cat# STA-325-5).
Oxiselect oxidative dna damage elisa
Manufactured by Cell Biolabs
Sourced in United States
The OxiSelect Oxidative DNA Damage ELISA is a quantitative assay designed to detect and measure the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, in various sample types.
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4 protocols using oxiselect oxidative dna damage elisa
1
Quantifying Oxidative Damage in Brain Regions
2
Quantifying Oxidative Stress Biomarkers
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Urine samples with sufficient volume were analyzed for 8-OHdG and F2-isoprostane at the New York University High Throughput Biology Laboratory (N = 2,465 samples, N = 618 individuals, and N = 1,287 samples, N = 522 individuals, respectively). 8-OHdG was quantified by competitive ELISA using the OxiSelect Oxidative DNA Damage ELISA (Cell Biolabs, San Diego, CA). Similarly, F2-isoprostane was measured with a competitive enzyme-linked immunoassay, the OxiSelect 8-iso-Prostaglandin F2α ELISA Kit (Cell Biolabs). All analyses were conducted in duplicate as directed by the manufacturer. Intra-assay CVs ranged from 4.6% to 11.1% for 8-OHdG and from 6.8% to 9.7% for F2-isoprostane. Inter-assay CVs ranged from 3.9% to 16.6% for 8-OHdG and from 14.2% to 15.9% for F2-isoprostane. Measures below the LOD were imputed by the LOD divided by the square root of 2 [50 ].
3
Oxidative Stress Biomarkers in Urine
4
Quantifying Oxidative DNA Damage by ELISA
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ROS levels were determined by measuring the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a ROS-mediated DNA damage by-product, with an oxidative DNA damage ELISA kit (OxiSelect Oxidative DNA Damage ELISA; Cell Biolabs, San Diego, CA). ELISA was performed according to the manufacturer’s instructions. Briefly, 100 μL of 8-OHdG–BSA conjugate in PBS was incubated overnight at 4°C in 96-well plates. After washing with distilled water, the plate was blocked with 200 μL of assay diluent for 1 h at room temperature. Samples and standards (50 μL) then were incubated for 10 min, followed by incubation for 1 h with 50 μL of anti–8-OHdG detection antibody. After washing, 100 μL of secondary antibody coupled with horseradish peroxidase was incubated for 1 h. After several washes, the peroxidase activity was detected by the addition of 100 μL of substrate solution and incubation for 5 min. The reaction was stopped with 100 μL of stop solution and measured at 450 nm on the Synergy H1 ELISA plate reader (BioTek, Winooski, VT). Data were standardized to total DNA concentration.
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