title: Use of pulsed-field gel electrophoresis to investigate factors influencing the measurement of DNA double-strand breaks in human brain tumour specimens.
abstract: We used pulsed-field gel electrophoresis combined with densitometry to investigate variables influencing the measurement of DNA double-strand breaks (dsb) in human brain tumour cells and frozen tissue. Our studies showed that this system worked best when we ran the gel at 60 V for 22-25 h at 18 degrees C with a pulse time of 15 s in 1xTris-acetic acid-EDTA buffer. Because the densitometric analysis worked well only when DNA concentrations were within a certain range, we developed a quick assay using a DNA-specific dye (TO-PRO-1) to determine concentrations before making agarose plugs. When we used our optimal procedure to measure dsb in six frozen human brain tumour specimens, we found that radiation-resistant tumours had significantly more initial dsb than did radiation-sensitive tumours. The number of residual dsb could not be ascertained because the process of freezing the specimens destroyed dsb repair ability.
authors.affiliation: Department of Neurological Surgery, School of Medicine, University of California, San Francisco 94143.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: D A
authors.initials: DA
authors.lastname: Delgado
authors.forename: D F
authors.initials: DF
authors.lastname: Deen
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics / Q000532:radiotherapy*; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D014407:Tumor Cells, Cultured
chemical_list: D004247:DNA