The EpiQuik 8-OHdG Direct DNA Damage Quantification Kit (Colorimetric) is a complete set of buffers and reagents optimized to detect and colorimetrically 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 in 3 hours and 45 minutes.
- High sensitivity, whose detection limit can be as low as 2 pg of 8-OHdG.
- High specificity when detecting only 8-OHdG without cross-reactivity with 8-OHdG analogs.
- 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 precision is highly correlated and close to HPLC or LC-MS analysis.
- Very convenient assay with direct use of DNA isolated from cells or tissues, without the need for digestion or hydrolysis of the DNA.
- Universal positive and negative controls are included, which are suitable for quantifying 8-OHdG of any species.
- The strip-well microplate format makes the assay flexible for manual or high-throughput testing.
- Simple, reliable, and consistent test conditions.
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 for its abundance but also for its mutagenic potential through G-to-T transversion mutations after DNA replication.
8-OHdG also participates in the epigenetic regulation of gene activation/repression by inhibiting the binding affinity of the 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 elevated 8-OHdG levels are closely related to exposure to harmful environmental factors such as ionizing radiation, industrial chemicals, air pollution, smoking, and cancer chemotherapy.
Various chromatography-based techniques, such as HPLC-ED and LC-MS, are used to detect 8-OHdG in tissues and cells. However, these methods are time-consuming and have low performance with high costs. Currently used competitive ELISA methods are also not conveniently applicable for the detection of 8-OHdG from cells/tissues because they are less accurate and have an inability to use intact DNA isolated from cells or tissues directly.
Principle and procedure
The EpiQuik 8-OHdG Direct DNA Damage Quantitation Kit (Colorimetric) contains all the reagents necessary for the quantification of oxidative DNA damage (8-OHdG). In this assay, DNA is bound to wells that are specifically treated to have a high affinity for DNA. 8-OHdG is detected by capture and detection antibodies. The detected signal is enhanced and then quantified colorimetrically by reading the absorbance on a microplate spectrophotometer. The amount of 8-OHdG is proportional to the DO intensity measured.
Starting materials and input quantity
The amount of DNA can range from 100 ng to 300 ng per reaction. An optimal amount is 300 ng per reaction. The starting DNA can be in water or in a buffer such as TE.
Safe and convenient
All necessary reagents, including negative controls and positive controls, for 8-OHdG quantitation, are conveniently packaged in the kit. Direct colorimetric quantification of DNA samples replaces outdated or inferior methods and eliminates the need for DNA digestion/denaturation, radioactivity, extraction, or chromatography.
Responsive, reliable, and practical
Based on its operating principle and microplate format, the kit can be practically and routinely used for any species in a variety of ways including cultured cells, fresh and frozen tissues, and paraffin-embedded tissues. To demonstrate the capabilities of the kit, it has been used successfully to quantify the content of 8-OHdG in the DNA of human-mouse kidney, liver, and brain tissues.