The EpiQuik™ Nitrosative (8-Nitroguanine) DNA/RNA Damage Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically detect and quantify nitrosative DNA/RNA damage (8-nitroguanine, 8-NG) status directly using DNA /RNA 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.
- High sensitivity with a detection limit as low as 2 pg of 8-NG.
- High specificity by detecting only 8-NG without cross-reactivity to 8-OhG, 8-OHdG, dG, guanine, within the indicated concentration range of the sample DNA.
- Direct detection of 8-NG using intact DNA or RNA, which eliminates interference from high molecular weight compounds, such as carbohydrates and proteins that are often seen in competitive 8-NG assays.
- Detection level is highly correlated with and close to HPLC or LC-MS analysis.
- Highly convenient assay with direct use of DNA or RNA isolated from cells or tissues, no need for DNA/RNA digestion or hydrolysis.
- Universal positive and negative controls are included, which are suitable for quantifying 8-NG from any species.
- Strip-well microplate format makes the assay flexible for manual or high throughput analysis.
- Simple, reliable, and consistent assay conditions.
8-nitroguanine (8-NG) is a mutagenic nitrative DNA/RNA lesion caused by reactive nitrogen species (RNS). As a modified nucleoside base, 8-NG is considered important because of its abundance and its mutagenic potential through G-to-T transversion mutations upon replication of DNA. 8-NG also participates in epigenetic regulation of gene activation/repression by causing breaks in DNA to form single-stranded DNA. It has also been demonstrated that increased concentrations of 8-NG are pathogenically linked to various inflammation-associated diseases, including cancer. Comparative urine/serum 8-NG assays mainly reflect the balance between nitrosative damage and the repair rate of the whole body. However, quantifying 8-NG content directly in different cells/tissues of normal/disease states allows tissue-specific nitrosative damage of DNA/RNA to be identified. Thus, this method provides more useful information about nitrosative damage-disease relationships to benefit disease diagnostics and therapeutics.
Principle & Procedure
This kit contains all reagents necessary for the quantification of nitrosative (8-nitroguanine) DNA/RNA. In this assay, DNA/RNA is bound to strip wells that are specifically treated to have a high nucleic acid binding affinity. 8-NG 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-NG is proportional to the OD intensity measured.
Starting Materials & Input Amount
The amount of total DNA or total RNA for each assay can be 100 ng to 300 ng. For optimal quantification, the input DNA and RNA amount should be 300 ng, as basal 8-NG is generally less than 0.01% of total DNA or RNA.
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