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ASTM International - ASTM E2186-02a(2016)

Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay

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Organization: ASTM International
Publication Date: 1 February 2016
Status: active
Page Count: 10
ICS Code (Microbiology in general): 07.100.01
significance And Use:

5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19).... View More

scope:

1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.

1.1.1 The Comet assay has been used to screen the genotoxicity of a variety of compounds on cells in vitro and in vivo (5-7), as well as to evaluate the dose-dependent anti-oxidant (protective) properties of various compounds (3, 8-11). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).

1.2 This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair rates (10), detection of the presence of photoactive DNA damaging compounds (14), or detection of specific DNA lesions (3, 18) .

1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

1.4 This guide is arranged as follows:

  Section
 
Scope 1
Referenced Documents 2
Terminology 3
Summary of Guide 4
Significance and Use 5
Equipment and Reagents 6
Assay Procedures 7
Treatment of Data 8
Reporting Data 9
Keywords 10
Annex Annex A1
References  

Document History

ASTM E2186-02a(2016)
February 1, 2016
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand...
March 1, 2010
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks...
April 10, 2003
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The...
January 10, 2002
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The...
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