Nested PCR for mtDNA-4977-bp deletion and comet assay for DNA damage - a combined method for radiosensitivity evaluation of tumor cells

Abstract

To identify an effective method of evaluating the radiosensitivity of human tumor cell lines in vitro, the present study adopted mtDNA-4977-bp deletion coupled with comet assay. The three human tumor cell lines applied were HepG2, EC-9706 and MCF-7. The surviving fraction (SF), ratio of the mtDNA-4977-bp deletion and DNA damage were detected by MTT assay, nested polymerase chain reaction (PCR) technique and comet assay, respectively. Clearly, lower SFs were found for the HepG2 and EC-9706 cells as compared with the MCF-7 cells following irradiation at doses of 2, 4 and 8 Gy, indicating a higher radiosensitivity for the HepG2 and EC-9706 cells. Additionally, no significant differences were identified in the mtDNA-4977-bp deletions found among HepG2, EC-9706 and MCF-7 cells by PCR following 1- or 4-Gy γ-ray irradiation, while increased deletion ratios of mtDNA-4977 bp were observed in HepG2 and EC-9706 cells following 8-Gy irradiation, in contrast to decreases in MCF-7 cells. The most notable differences among these three tumor cell lines were observed by comet assay following 8–Gy γ-ray irradiation. A combined method of nested PCR and comet assay, therefore, is the most effective and accurate method in evaluating the radiosensitivity of tumor cells.

Radioprotective effect of lactoferrin in mice exposed to sublethal X‑ray irradiation

“…The comet assay, which detects DNA damage, has been widely used in radiation biology (37)(38)(39)(40). The comet assay is a rapid and sensitive microdosimetric technique, particularly useful in radiation accidents (41).…”

Evaluation on the genetic instability detecting methods for rapid diagnose of Fanconi anemia used in the undeveloped areas of China

“…The SCGE assay was performed using an adapted method described before; 0.5 mL peripheral blood was carefully layered onto the lymphocyte‐separating medium in a 5‐mL centrifugation tube. After centrifugation for 5 minutes at 650 g , the gray lymphocytes between the blood plasma and the lymphoprep layer were carefully transferred into a centrifugation tube.…”

“…The alkaline SCGE assay is a sensitive and simple method for evaluating DNA damage and has been used for the detection of the DNA damage in patients with breast cancer or individuals who have been accidentally exposed to ionizing radiation, and the diagnosis of patients with FA. [18][19][20] Here, SCGE was used for the distinction of patients with FA and patients with AAA. The comet images of lymphocyte nuclei derived from patients with FA, with AAA, and the controls showed the intensity of DNA damage ( Figure 3A-C).…”

Ginger Oleoresin Alleviated γ‐Ray Irradiation‐Induced Reactive Oxygen Species via the Nrf2 Protective Response in Human Mesenchymal Stem Cells

“…The DNA damage in an individual cell was measured by using the gel electrophoresis-based comet assay [ 33 ]. Under alkaline conditions, the negatively charged DNA supercoils with broken ends were able to migrate toward the anode during electrophoresis.…”

“…For comet assay, hMSCs were pretreated with 10 −4 g/mL ginger oleoresin and cultured for 2 h followed by exposure to γ -rays at the Irradiation Center. After irradiation, the cells were subsequently incubated for 24 h. The comet assay was performed as described before [ 33 ]. Briefly, the treated cell samples were applied to prepare single-cell suspensions using trypsin disaggregation.…”