Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Al-Salmani, Kamla; Abbas, Hussein H. K.; Schulpen, Sjors H.W.; Karbaschi, Mahsa; Abdalla, Intisar; Bowman, Karen J.; So, Kwok K.; Evans, Mark D.; Jones, George D.D.; Godschalk, Roger; Cooke, Marcus S.

doi:10.1016/j.freeradbiomed.2011.05.020

Cited by 74 publications

(67 citation statements)

References 23 publications

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“…The comet assay for PBMC was performed adjusted to the method described by Azqueta et al [38] in a 12-minigel format, as described by the same author [39]. The comet assay for whole blood was performed according to Al-Salmani et al [40] with slight modifications. Stored whole blood was thawed quickly, 10 μl whole blood was mixed with 200 μl 1%-agarose solution and 5 μl were pipetted on the respective spot of the 12-minigel slide.…”

Section: Methodsmentioning

confidence: 99%

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

et al. 2016

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BackgroundDiabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.MethodsFemale T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.ResultsNo significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.ConclusionBMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria.

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Section: Methodsmentioning

confidence: 99%

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

et al. 2016

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“…It is possible (and more usual) to isolate mononuclear cells (mostly lymphocytes) by differential centrifugation. Recently, a simple procedure for isolating the complete leukocyte population by simple centrifugation from thawed frozen blood, in which the red cells have lysed, has been described [33][34]. The isolated white cells can then be challenged with H 2 O 2 to assess antioxidant resistance by the yield of DNA breaks [34].…”

Section: Discussionmentioning

confidence: 99%

Atmospheric pollution in Kosovo is associated with increased DNA damage in the human population

Alija¹,

Asllani²,

Bajraktari³

et al. 2016

Biomonitoring

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Abstract:In order to assess DNA damage associated with exposure to environmental pollution in two polluted sites and one control site in Kosovo, whole blood samples were collected from volunteers in two polluted areas (Kastriot/ Obiliq - lignite-based power plants and lignite mines - and Drenas/Gllogovc - Ferronikeli smelting plant) as well as from Peja, representing an unpolluted area. White blood cells were isolated, and DNA damage was analyzed by the alkaline comet assay. Significantly higher levels of DNA damage (strand breaks) were found in white blood cells from subjects living in the polluted areas compared with residents of the unpolluted city, indicating a potential threat to human health.

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“…Although this is the most frequent procedure for sample preservation, Al-Salmani et al [ 42 ] showed that whole blood can be preserved in small aliquots (of approximately 250 μL) without cryopreservative for up to 1 month at −80 °C without artifactual formation of DNA damage.…”

Section: Sample Preservationmentioning

confidence: 99%

The Comet Assay In Vivo in Humans

Costa

Teixeira

2014

Genotoxicity and DNA Repair

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In the last decades, the comet assay has been used as a molecular biomarker to detect DNA damage in several human biomonitoring studies. Nevertheless, many of these studies are often inconclusive or offer poor quality data due to study design constraints. This chapter describes not only the experimental protocol to carry the alkaline version of the comet assay in human cells but also considers many of the obstacles that the researcher faces while carrying a human biomonitoring study, such as sample size, sample type, sample collection, statistics, and result communication. Understanding assay limitations, both experimental and biological, is essential to improve both data quality and data relevance obtained and to guarantee that comet assay continues to provide enhanced reliability as a biomarker in human biomonitoring studies.Key words Comet assay , Human biomonitoring , Exposure , Lymphocytes , Whole blood Comet Assay for Human BiomonitoringComet assay (also designated of single cell gel electrophoresis) is a rapid, simple, visual, and sensitive method to detect DNA breakage in eukaryotic cells [ 1 ].Briefl y, cells are embedded in agarose on a microscope slide, lysed with detergent to remove cell and nuclear membranes, and treated with a high salt solution. Nucleoids are formed, containing non-nucleosomal but still supercoiled DNA. Any breaks present in the DNA cause the supercoiling to relax locally and loops of DNA are then free to extend toward the anode during electrophoresis. At the end of this procedure, damaged cells will resemble comets (hence the name comet assay ) while non-damaged cells maintain their round appearance. Comets are viewed by fl uorescence microscopy after staining with a suitable fl uorescent DNA-binding dye [ 2 ].The pH of unwinding solution is responsible for differences in method sensitivity, as the conversion of alkali-labile sites to strand breaks is a process dependent on the pH and time of alkaline treatment. Under alkaline (pH > 13) conditions, the assay potentially

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Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Cited by 74 publications

References 23 publications

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

Atmospheric pollution in Kosovo is associated with increased DNA damage in the human population

The Comet Assay In Vivo in Humans

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