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Novel high throughput method for comet slide processing

Researchers from the Department of Environmental and Occupational Health at Florida International University have published a paper in Scientific Report which details a novel, patent pending, method for the high-throughput (HT) processing of slides for the comet assay.

Single cell gel electrophoresis, or the comet assay, continues to attract growing interest as a tool to study the formation and repair of DNA damage, both in vitro and in vivo studies, as markers of genotoxicity.

Many people believe comet slide sample preparation and processing (e.g. lysis, electrophoresis, washing, and staining steps) is time consuming and laborious.  To address this obstacle, the scientists have developed a novel method by which multiple slides may be manipulated.  The slides undergo processing and electrophoresis in batches of 25 (rather than individually).  Importantly, this method uses standard, conventional microscope comet slides, so there is no need to major changes to the assay – just a novel tank design and rack.  For full experimental details, please refer to the original publication.

The simultaneous manipulation of up to 25 comet assay slides is achieved by using a polyoxymethylene rack, which was termed the HT rack.  The same rack allowed electrophoresis to be performed with the slides held, lengthwise, in a vertical orientation – this significantly decreases the footprint of the tank, as it also has integrated chilling.  In order to assess the effect of performing electrophoresis on slides in the vertical orientation in the HT rack, the level of DNA damage and the quality of comets were compared by performing the alkaline comet assay in the conventional, horizontal orientation. All slides were then observed and scored by fluorescent microscopy (50 cells per gel; 100 cells per treatment), and percentage tail DNA of the comets was recorded, using Comet Assay IV analysis software, version 4.2 (Perceptive Instruments, Suffolk, UK).

The researchers believe that this method decreases assay time by 60%.  Also, the electrophoresis tank used in this study has a substantially smaller footprint and is able to generate the more uniform orientation of gels during electrophoresis.

This HT variant of the comet assay:

  • increases the number of samples analysed
  • decreases assay time
  • decreases number of individual slide manipulations
  • decreases reagent requirements
  • decreases the risk of damage to slides
  • decreases the amount of bench space required for the electrophoresis tank.  (The researchers also point out that potentially, multiple electrophoresis tanks can be run simultaneously from a single power source.)

The scientists believe that this novel approach represents a significant advance on the current, conventional, comet assay procedure.  Furthermore, the approach is complemented by a novel, chilling plate that allows rapid solidification of the cell-containing gels on the microscope slides.  The chilling plate conveniently chills 26 slides simultaneously, without the need for ice, which prevents condensation and improves slide handling.

The apparatus described here (HT comet assay and chilling plate) were developed in collaboration with Cleaver Scientific Ltd (Rugby, UK), and for full explanation of the experiments and equipment used in this investigation, please refer to the original publication: Novel method for the high-throughput processing of slides for the comet assay, Karbaschi M, Cooke MS. Scientific Reports 4, Article number: 7200 (2014).

Thank-you to Marcus Cooke and Mahsa Karbaschi for the assistance in writing this article and for allowing us to use their images. The Cooke Lab at FIU purchased Comet Assay IV in 2014 and are using the system for multiple investigations and research projects.  Marcus Cooke, Head of Dept. Environmental & Occupational Health, had used Comet Assay IV at a previous position at the University of Leicester and was pleased with the performance of the system.

Cooke and Karbaschi are not new to the development of improvement to the comet assay.  In 2012, they were part of a team that described how a pin-prick (5 µL) of whole blood could be used directly in the comet assay, without the need for the time-consuming isolation of peripheral blood mononuclear cells.  Additionally, they showed that the same small volumes of blood (up to 250 µL) could be stored at -80 °C for up to one month, without the need for a cryopreservative, and be subsequently thawed and used in the comet assay with no formation of artefactual DNA damage.  Combined, these are significant advances for using the comet assay for biomonitoring. See: Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood. Al-Salmani, K., Abbas, HHK., Schulpen, S., Karbaschi, M., Abdalla, I., Bowman, KJ., So, KK., Evans, MD., Jones, GDD., Godschalk, R. and Cooke, MS.  (2011)  Free Radic. Biol. Med., 51, 719-725.

 

 

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