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EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric)

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For quantitating 8OHdG for oxidative DNA damage in DNA samples

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Suggested Workflow
DNA Isolation
 
 
DNA Damage & Repair
 
Schematic procedure for the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric).
8-OHdG standard control was added into the assay wells at different concentrations and then measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit.
Oligos containing 8-OHdG, or 8-OHG or only dG were added into the assay wells at the different concentrations and then measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric).
Percentage of 8-OHdG in different tissues measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit.
Input Type: DNA
Research Area: DNA Damage & Repair
Target Application: Amount Quantitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-6003-4848 assays $379.00 
P-6003-9696 assays $599.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically detect and quantify oxidative DNA damage (8-OHdG) directly using DNA isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues,  and body fluid samples. The kit has the following advantages and features:

  • Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 45 minutes.
  • High sensitivity, of which the detection limit can be as low as 2 pg of 8-OHdG (see Fig. 2).
  • High specificity by detecting only 8-OHdG without cross-reactivity to 8-OHdG analogues (see Fig. 3).
  • Direct detection of 8-OHdG using intact DNA, which eliminates interference from high molecular weight compounds, such as carbohydrates and proteins that are often seen in competitive 8-OHdG assays.
  • Detection accuracy is highly correlated with and close to HPLC or LC-MS analysis (see Table 1).
  • Highly convenient assay with direct use of DNA isolated from cells or tissues, without the need for DNA digestion or hydrolysis.
  • Universal positive and negative controls are included, which are suitable for quantifying 8-OHdG from any species.
  • Strip-well microplate format makes the assay flexible for manual or high throughput analysis.
  • Simple, reliable, and consistent assay conditions.

Background Information
8-hydroxy-2’-deoxyguanosine (8-OHdG or 8-oxo-dG) is an oxidized derivative of deoxyguanosine and is generated by hydroxyl radicals, singlet oxygen, and one-electron oxidants in cellular DNA. As a modified nucleoside base, 8-OHdG is considered important not only because of its abundance but also because of its mutagenic potential through G-to-T transversion mutations upon replication of DNA. 8-OHdG also participates in epigenetic regulation of gene activation/repression by inhibiting the binding affinity of MBD protein to the CpG sites of DNA. Currently, 8-OHdG is widely accepted as a sensitive marker of oxidative DNA damage and oxidative stress. Evidence shows that increased levels of 8-OHdG are closely correlated with exposure to harmful environmental factors such as ionizing radiation, industrial chemicals, air pollution, cigarette smoking, and cancer chemotherapy.

Several chromatography-based techniques such as HPLC-ED and LC-MS are used for detecting 8-OHdG in tissues and cells. However these methods are time consuming and have low throughput with high costs. The currently used competitive ELISA methods are also not conveniently applicable for cell/tissue 8-OHdG detection because they are less accurate and have an inability to use intact DNA isolated from cells or tissues directly.

Principle & Procedure
The EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) contains all reagents necessary for the quantification of Oxidative DNA damage (8-OHdG). In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. 8-OHdG is detected using capture and detection antibodies. The detected signal is enhanced and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The amount of 8-OHdG is proportional to the OD intensity measured.

Starting Materials & Input Amount
DNA amount can range from 100 ng to 300 ng per reaction. An optimal amount is 300 ng per reaction. Starting DNA may be in water or in a buffer such as TE.

Safe and Convenient
All the necessary reagents, including negative controls and positive controls, for the quantification of 8-OHdG are conveniently packaged in the kit. The direct colorimetric quantification of DNA samples replaces obsolete or inferior methods and eliminates the need for DNA digestion/denaturation, radioactivity, extraction, or chromatography.

Responsive, Reliable, and Practical
Based on its working principle and the microplate format, the kit can be practically and routinely used for any species in a variety of forms including cultured cells, fresh and frozen tissues, and paraffin-embedded tissues. To demonstrate the capabilities of the kit, it has been successfully used for quantifying the content of 8-OHdG in DNA from human kidney, liver, and mouse brain tissues. The percentage of 8-OHdG measured by the kit is similar and comparable to that detected by HPLC methods (see Table 1 and Fig. 4). 

Fig. 1. Schematic procedure for the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric).


Fig. 2. 8-OHdG standard control was added into the assay wells at different concentrations and then measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit.

 
Fig. 3. Oligos containing 8-OHdG, or 8-OHG or only dG were added into the assay wells at the different concentrations and then measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric). 

 
Fig. 4. Percentage of 8-OHdG in different tissues measured with the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit. 

 
Table 1. Comparison of the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit and HPLC-based methods for the quantification of 8-OHdG, in two DNA sample types.

 

Product Components

WB (10X Wash Buffer)
BS (Binding Solution) 
NC (Negative Control , 10 µg/ml)
PC (Positive Control, 8-OHdG 1 µg/ml)
CA (Capture Antibody, 100 X) 
DA (Detection Antibody, 1000 X)
ES (Enhancer Solution)
DS (Developer Solution)
SS (Stop Solution)
8-Well Assay Strips (With Frame)
User Guide

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations

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Loch-Caruso R et. al. (November 2018). Trichloroethylene exposure in mid-pregnancy decreased fetal weight and increased placental markers of oxidative stress in rats. Reprod Toxicol. 83:38-45.

Maria Moros et. al. (June 2018). Light-triggered modulation of cell antioxidant defense by polymer semiconducting nanoparticles in a model organism MRS Communications. :1-8.

Smolensky D et. al. (May 2018). High-Polyphenol Sorghum Bran Extract Inhibits Cancer Cell Growth Through ROS Induction, Cell Cycle Arrest, and Apoptosis. J Med Food.

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Esplugas R et. al. (February 2018). Renal and hepatic effects following neonatal exposure to low doses of Bisphenol-A and <sup>137</sup>Cs. Food Chem Toxicol. 114:270-277.

Ceylan D et. al. (December 2017). DNA redox modulations and global DNA methylation in Bipolar Disorder: Effects of sex, smoking and illness state Psychiatry Research.

Haque S et. al. (November 2017). Monocyte-derived exosomes upon exposure to cigarette smoke condensate alter their characteristics and show protective effect against cytotoxicity and HIV-1 replication. Sci Rep. 7(1):16120.

Tatsuki Kunoh et. al. (October 2017). Green Synthesis of Gold Nanoparticles Coupled with Nucleic Acid Oxidation ACS Sustainable Chemistry & Engineering.

Zhenqiang You et. al. (September 2017). Modulatory Effect of Fermented Papaya Extracts on Mammary Gland Hyperplasia Induced by Estrogen and Progestin in Female Rats Oxidative Medicine and Cellular Longevity. 2017

Hara K et. al. (September 2017). Scavenging of reactive oxygen species by astaxanthin inhibits epithelial-mesenchymal transition in high glucose-stimulated mesothelial cells. PLoS One. 12(9):e0184332.

Afshari P et. al. (September 2017). Reduced Slc1a1 expression is associated with neuroinflammation and impaired sensorimotor gating and cognitive performance in mice: Implications for schizophrenia. PLoS One. 12(9):e0183854.

E. Tvrda et. al. (July 2017). Antioxidant effects of lycopene on bovine sperm survival and oxidative profile following cryopreservation Veterinarni Medicina. 62(8):429–436.

Bellés M et. al. (March 2017). Environmental exposure to low-doses of ionizing radiation. Effects on early nephrotoxicity in mice. Environ Res. 156:291-296.

Noguera JC et. al. (March 2017). Interacting effects of early dietary conditions and reproductive effort on the oxidative costs of reproduction. PeerJ. 5:e3094.

Ng CT et. al. (February 2017). Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and <i>Drosophila melanogaster</i>. Int J Nanomedicine. 12:1621-1637.

Tasaki E et. al. (January 2017). An Efficient Antioxidant System in a Long-Lived Termite Queen. PLoS One. 12(1):e0167412.

Daniel Nettle et. al. (December 2016). Early-life adversity accelerates cellular ageing and affects adult inflammation: Experimental evidence from the European starling Scientific Reports. Dec. 2016

Szmidt M et. al. (July 2016). Toxicity of different forms of graphene in a chicken embryo model. Environ Sci Pollut Res Int.

Adamczyk J et. al. (June 2016). Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts. Cell Stress Chaperones.

Yost AD et. al. (October 2015). Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage. PLoS One. 10(10):e0139903.

Deregowska A et. al. (October 2015). Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts. Oncotarget. 6(31):30650-63.

Philbrook NA et. al. (October 2015). Benzoquinone toxicity is not prevented by sulforaphane in CD-1 mouse fetal liver cells. J Appl Toxicol.

Shimizu R et. al. (April 2015). Cholangiocyte senescence caused by lysophosphatidylcholine as a potential implication in carcinogenesis. J Hepatobiliary Pancreat Sci.

Nocchi L et. al. (June 2014). Induction of oxidative stress causes functional alterations in mouse urothelium via a TRPM8-mediated mechanism: implications for aging. Aging Cell. 13(3):540-50.

Di Nunzio M et. al. (November 2013). Counteraction of oxidative damage by pomegranate juice: influence of the cultivar. J Sci Food Agric. 93(14):3565-73.

Sobinoff AP et. al. (September 2013). Scrambled and fried: cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress. Toxicol Appl Pharmacol. 271(2):156-67.

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