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Some causes of inter-laboratory variation in the results of comet assay. 2014

Nikolai P Sirota, and Aliy K Zhanataev, and Elena A Kuznetsova, and Eugenii P Khizhnyak, and Elena A Anisina, and Andrei D Durnev
Institute of Theoretical and Experimental Biophysics of Russian Academy of Sciences, Pushchino, Russia. Electronic address: sirota@iteb.ru.

We performed an inter-laboratory study to determine the variation of comet assay results and to identify its possible reasons. An exchange of slides between Labs in different stages of the comet assay protocol was performed. Because identical slides, durations of alkali treatment and electrophoresis, and similar electric field strengths (2.0 V/cm and 2.14 V/cm) were used, we concluded that the observed inter-laboratory difference in the results is directly associated with the electrophoresis step. In Lab 1, mouse bone marrow cells were exposed to methyl methanesulfonate at concentrations of 10, 25 and 50 μM for 3 h at 37 °C. In Lab 2, cells the same as in Lab 1 were immobilized in LMA on slides and exposed to X-rays at doses of 3-8 Gy. We found that the transportation of slides after lysis or electrophoresis step, as well as different dyes used for scoring did not produce any significant effect on the results. No substantial difference in the data was also revealed when various software packages were used for image analysis. The temperature of the alkaline solution was shown to increase during electrophoresis and, besides, the temperature heterogeneity of the solution took place in the area of the platform, with a maximum in the middle of the chamber. The temperature heterogeneity could affect the rate of conversion of alkali labile sites into single stranded breaks. Thus, it was clearly indicated that real temperature variations during the alkali treatment and electrophoresis were an essential factor in the variability of the results between our Labs.

UI MeSH Term Description Entries
D007753 Laboratories Facilities equipped to carry out investigative procedures. Laboratory
D008741 Methyl Methanesulfonate An alkylating agent in cancer therapy that may also act as a mutagen by interfering with and causing damage to DNA. Methylmethane Sulfonate,Dimethylsulfonate,Mesilate, Methyl,Methyl Mesylate,Methyl Methylenesulfonate,Methylmesilate,Mesylate, Methyl,Methanesulfonate, Methyl,Methyl Mesilate
D008810 Mice, Inbred C57BL One of the first INBRED MOUSE STRAINS to be sequenced. This strain is commonly used as genetic background for transgenic mouse models. Refractory to many tumors, this strain is also preferred model for studying role of genetic variations in development of diseases. Mice, C57BL,Mouse, C57BL,Mouse, Inbred C57BL,C57BL Mice,C57BL Mice, Inbred,C57BL Mouse,C57BL Mouse, Inbred,Inbred C57BL Mice,Inbred C57BL Mouse
D001854 Bone Marrow Cells Cells contained in the bone marrow including fat cells (see ADIPOCYTES); STROMAL CELLS; MEGAKARYOCYTES; and the immediate precursors of most blood cells. Bone Marrow Cell,Cell, Bone Marrow,Cells, Bone Marrow,Marrow Cell, Bone,Marrow Cells, Bone
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D004249 DNA Damage Injuries to DNA that introduce deviations from its normal, intact structure and which may, if left unrepaired, result in a MUTATION or a block of DNA REPLICATION. These deviations may be caused by physical or chemical agents and occur by natural or unnatural, introduced circumstances. They include the introduction of illegitimate bases during replication or by deamination or other modification of bases; the loss of a base from the DNA backbone leaving an abasic site; single-strand breaks; double strand breaks; and intrastrand (PYRIMIDINE DIMERS) or interstrand crosslinking. Damage can often be repaired (DNA REPAIR). If the damage is extensive, it can induce APOPTOSIS. DNA Injury,DNA Lesion,DNA Lesions,Genotoxic Stress,Stress, Genotoxic,Injury, DNA,DNA Injuries
D004305 Dose-Response Relationship, Drug The relationship between the dose of an administered drug and the response of the organism to the drug. Dose Response Relationship, Drug,Dose-Response Relationships, Drug,Drug Dose-Response Relationship,Drug Dose-Response Relationships,Relationship, Drug Dose-Response,Relationships, Drug Dose-Response
D004307 Dose-Response Relationship, Radiation The relationship between the dose of administered radiation and the response of the organism or tissue to the radiation. Dose Response Relationship, Radiation,Dose-Response Relationships, Radiation,Radiation Dose-Response Relationship,Radiation Dose-Response Relationships,Relationship, Radiation Dose-Response,Relationships, Radiation Dose-Response
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D014965 X-Rays Penetrating electromagnetic radiation emitted when the inner orbital electrons of an atom are excited and release radiant energy. X-ray wavelengths range from 1 pm to 10 nm. Hard X-rays are the higher energy, shorter wavelength X-rays. Soft x-rays or Grenz rays are less energetic and longer in wavelength. The short wavelength end of the X-ray spectrum overlaps the GAMMA RAYS wavelength range. The distinction between gamma rays and X-rays is based on their radiation source. Grenz Ray,Grenz Rays,Roentgen Ray,Roentgen Rays,X Ray,X-Ray,Xray,Radiation, X,X-Radiation,Xrays,Ray, Grenz,Ray, Roentgen,Ray, X,Rays, Grenz,Rays, Roentgen,Rays, X,X Radiation,X Rays,X-Radiations

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