Yearly Archives

2 Articles

Posted by ivocabral ivocabral on

Video – Comet assay, step by step

The Norwegian Institute for Air Research (NILU), coordinator of RiskGONE, created a short video showing the main steps in conducting a comet assay, in order to assess the toxicity of nanomaterials.

One of the aims of the RiskGONE project is the verification, optimisation and development of methods for the characterization and human and eco-toxicological hazard assessment of ENMs (engineered nanomaterials). This is done through interlaboratory round robin exercises and training on selected methods. One of these methods is the Comet assay (single cell gel electrophoresis), a simple method used for measuring DNA damage in eukaryotic cells. The method is widely used for detection of strand breaks as well as specific DNA lesions, such as oxidized purines and pyrimidines, and is considered a useful method for genotoxicity testing in vitro as well as in vivo.

NILU provided RiskGONE partners with training on this method at a course that took place in February 2020, and in March 2021, through a practical online course/video learning due to the COVID-related restrictions. The video available below is part of the training material used in this course.

How the Comet Assay works

After exposure to the compound of interest (in this case, ENM), the cells are embedded in agarose on a microscope slide. After treatment with a detergent solution, membranes, cytoplasm, and most of the soluble cell contents are dissolved, and the DNA nucleoids are freed. The nucleoids are then subjected to an electrophoretic field, which makes the negatively charged DNA migrate towards the positive electrode-anode. DNA in the nucleoids is very compact and its movement is limited, but if a break is present in the DNA strands, the DNA loop is free to extend under the electrophoretic field and move towards the anode. When DNA is stained with specific dyes and examined microscopically, images resembling comets are seen; the comet tail consists of loops of DNA that, due to the presence of damage (strand break), have moved out from the nucleoid (comet head). The amount of DNA in the tail reflects the number of breaks in the DNA.

The standard comet assay measures single- and double-strand breaks. A modified version of the assay by inclusion of lesion-specific enzymes can detect specific DNA lesions, such as oxidized purines using formamidopyrimidine DNA glycosylase (Fpg).

Posted by ivocabral ivocabral on

Comet Assay – Practical online course/video training by NILU

Comet Assay – Practical online course/video training by NILU

Making the gel drops: cells embedded in agarose are put on slides

Dates: 15th-19th March 2021

Location: remote/virtual

The Norwegian Institute for Air Research (NILU), coordinator of RiskGONE, is providing a practical online course/video learning on the Comet assay.

This training workshop represents the second edition of a series of trainings to be organised as part of RiskGONE`s training activities. The first edition of the course was physically held at NILU`s premises in Kjeller, Norway, on 17th-20th February 2020. Then, RiskGONE project partners joined the course from Swansea University, the University of Birmingham, and the University of Bergen, and they were trained on different assays for use with engineered nanomaterials.

This course welcomes participants from H2020 projects Twinalt and VISION, besides RiskGONE and the H2020-NMBP-13 network.

Due to the restrictions now imposed by the COVID-19 pandemic, the second edition of the course was re-arranged to a remote format. The course will take place for 5 days.

On the first day, the background and principles of the assay will be introduced in a series of lectures.

On the second day, the technical aspects of the assay will be presented. Part of the day will be dedicated to the planning of a real experiment. Trainees will have the opportunity to perform themselves in their own laboratory, with the constant support of the trainers.

On the following days, a hands-on experiment will be performed. In the morning, the daily work will be presented thought videos created at NILU, in which the trainers show how the experiment is performed step-by-step. In this phase, trainees will have the opportunity to interactive live with the trainers, ask questions and discuss the technical aspects of the experiment. In the afternoon, after viewing the videos, trainees will have the possibility to physically train on the experiment in their laboratory. All along, trainers will be available online to support trainees if needed.

Training materials, including the step-by-step videos, will be shared among all project partners and might also form the basis of teaching and training material to be used beyond the project course.

The training’s agenda can be accessed here