DNA Damage Quantification in TP53 Gene

Total genomic DNA was isolated according to Mullenbach et al. [53 (link)], and following previously described steps to reduce artifactual DNA damage [54 (link)]. DNA damage was quantified using two distinct methods: a PCR based assay (q-PADDA) which detects many types of DNA damage with high sensitivity [52 (link), 54 (link)], and a colorimetric based assay (HT 8-oxo-dG ELISA Kit II, Trevigen, MD) which detects exclusively 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) lesions. 8-oxo-dG is one of the major products of DNA oxidation. The primer-anchored DNA damage detection assay (q-PADDA) was performed as we previously described [52 (link)]. We chose to quantify DNA damage within the transcribed strand (TS) and non-transcribed strand (NTS) of TP53 (commonly referred as p53), because p53 is the most frequently mutated gene in human cancer [55 (link)] and is mutated in nearly all smoking related cancers.[56 (link), 57 (link)] For DNA damage quantification, as well as for all other analysis described below, we performed three independent experiments, each with 3–6 technical replicates.

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Ganapathy V., Manyanga J., Brame L., McGuire D., Sadhasivam B., Floyd E., Rubenstein D.A., Ramachandran I., Wagener T, & Queimado L. (2017). Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage. PLoS ONE, 12(5), e0177780.

Publication 2017

HT 8-oxo-dG ELISA Kit II

8 oxo dg Assay Cancer gene Cancers Colorimetric Dna damage Elisa Genomic Human Primer Sensitivity Tp53 Types dna

Corresponding Organization : University of Oklahoma Health Sciences Center

Other organizations : Stony Brook University, University of Madras

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Variable analysis

independent variables

DNA isolation method (Mullenbach et al. protocol)

dependent variables

DNA damage quantification using q-PADDA assay
DNA damage quantification using 8-oxo-dG ELISA assay

control variables

Transcribed strand (TS) and non-transcribed strand (NTS) of TP53 gene
Technical replicates (3-6) for each experiment

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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