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New In Vitro 8-Hydroxydeoxy Guanosine Assay - 8 OHDG Test

General Aim

• The OHDG test experiment aims at visualizing the response of protein recruitment to DNA damage sites. 
• DNA double strand breaks (DSBs) induce the hydroxylation of 2’-deoxyGuanosine to form 8-Hydroxy, 2’-deoxy Guanosine (8-OHdG), and accumulate 8-OHdG which can then be detected as DNA damage foci. .
• The detection of foci by immunostaining with antibodies that recognize 8-OHdG is an indicator of DSBs presence. 
• This 8 OHDG assay will describe the measurement of 8-OHdG immunostaining using a fluorescent microscope.

Method of 8 OHDG Test

In Vitro Immunofluorescence Assay for detection of 8-OHdG and DNA Damage.

Theoretical Background / Context

• In genetics, genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. 
• While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. 
• The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. 
• The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. 
• Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Principle of Work

• High levels of reactive oxygen species (ROS) might result in structural and/or genetic changes in cells modulating the initial steps of carcinogenesis. 
• As the accumulation of ROS and subsequent oxidative damage is commonly observed and widely studied in inflammatory diseases, thus, inflammation-induced ROS are linked to mutations in proto-oncogenes and tumour suppressor genes during cancer progression. 

• 8-Hydroxy-2’-deoxy Guanosine (8-OHdG) is a single nucleotide base lesion and is recognized as a biomarker of  oxidative DNA damage mechanism. 
• In the 8 OHDG test experiment, the detection of 8-OHdG is performed with a wide range of methodologies including immunofluorescence detection labelling which has certain benefits such as being performed with low numbers of cells in comparison to the analytical methods, while more cells are required to isolate relatively higher amounts of DNA for reliable quantitation of the damaged base. 
• When one hundred cells are counted per sample in the 8 ohdg test, defined object criteria are applied to determine parameters such as number of cells analyzed, number of foci per cell, their sizes, volumes and the number of cells having or not having foci using 8 ohdg antibody.