Introduction

The single cell gel electrophoresis assay that is also regarded as the comet assay, entails a flexible method used to measure varied classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. Despite this, its utility has been limited by the difficulties with reproducibility and limited throughput, more so for epidemiological and clinical studies. There has been an establishment of varied high throughput comet assay methods to help take care of these limitations. Among these methods there is:

1. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification

A microarray comet assay was created. Using a micrometer scale array of cells helps in increasing the number of analyzable comets per cm2 and this makes it possible for automated imaging and analysis. The platform also has a favorable compatibility with standard 96- and 24-well plate formats. The sensitivity of the microarray comet assay has been reviewed in this respect as well as its consistency. As a result, it was determined that H2O2-induced DNA damage that is found in the human lympoblastoid cells has a linear detection range between 30 and 100 μM. The inter-sample coefficient of variance was determined to be between 5 to 10% while working with this range.

Determining the statistically considerable DNA doses that would be sufficient for induction within this range required only 20 comets. There was also an evaluation of experiment to experiment and sample to sample variations. The findings indicated that the coefficient of variation was lower compared to the traditional comet assay’s reported figures in both conditions.

There has also been an illumination that proves the assay can be performed by use a 4x objective (rather than the standard 10x objective for the traditional assay). It becomes possible to capture more than 50 analyzable comets while combining the adjustment with the microarray format in a single image. This makes it to be automatically analyzed by use of in-house software. In general, there is an increase in throughput with more than 100 fold when a comparison is made with the traditional assay.

These results are an indication of advancement in the technology for comet assay since there is improvement in sensitivity, robustness and throughput. The advancement makes it possible for the enhancement of larger scale epidemiological and clinical studies. There is the possibility of increasing throughput by two orders of magnitude.

Limitation

Through the CometChip, it is possible to overcome the predicament of handling more than one sample in parallel; both in terms of required time and physically. It has also become easy to overcome sample to sample variability, which become worse when there is a need to process more samples in parallel. These are key limitations associated with the existing comet assay. Using a large glass slide that contains multiple minigels on the surface has been touted to advance reproducibility and throughput by published studies.

Advantage

The CometChip tends to integrate this advance using a 96 well plate, which is easy to handle. It is easier to process 96 samples that are on a single CometChip compared to handling 96 slides that has proved to exceed the capacity that most researchers can handle.

The CometChip helps to avoid problems that come with overlapping comets and this helps in settling wells that have a uniform depth. This aspect is complimented by its ability of being easier to handle. These aspects make it possible for CometChip to image around 63 analyzable comets in one image, and this reduces sample to sample and comet to comet variation.

2. High Throughput Comet System COMPAC- 50

COMPAC-50 has come out as a high throughput electrophoresis system that was developed at the University of Leicester in collaboration with the Oxidation Stress group. The electrophoresis system is exclusively available through Cleaver Scientific. It is meant to perform the Comet Assay, which is also referred to as Single Cell Gel Electrophoresis. There is a patent pending and it is unique since the design used enables two carriers to hold an overall of 50 slides (25 per carrier) in a vertical stratified orientation. This aspect ensures that the method has an edge over the Conventional Assay system. Among the advantages is that it produces a small footprint, highly compact system that optimizes the electrophoretic conditions through its economical use of buffer. Another advantage is that it reduces the assay time since it allows the holding of 25 slides from each side within a rack making it easy for all the slides to be processed within one batch. In addition to this being favorable for electrophoresis, it is also helpful in the neutralization, lysis, staining and washing steps of the Comet Assay. Each batch of the slides has the ability of being treated during every step by use of the four ebony acrylic staining dishes. COMPAC-50 tends to benefit from the ceramic cooling base with sliding draw to enable accommodation of a cool pack as it enhances performance. The cooling base is frozen before use to ensure that it maintains an optimal buffer temperature. This makes COMPAC-50 the pioneer of a new range of innovative systems that is developed for swift high throughput single sell gel electrophoresis.

The use of COMPAC -50 helps to reduce the assay time by 60%, and also gains from an electrophoresis tank that has a significantly smaller footprint coupled with uniform orientation of gels during electrophoresis. The high throughput variant that is associated with the comet assay elevates the number of samples analyzed, number of each slide manipulations, decrease assay time, risk of damage to slides and reagent requirements. Laboratories also tend to benefit from the compact nature of the electrophoresis tank at a premium bench space. The novel approach in use works as an important advance to the existing comet assay procedure.

Limitation

The throughput of the comet assay has two main limitations. Among them is the Scoring of comets. It is more of a manual determination of the extent of DNA damage in fifty cells of every gel and two gels for every treatment using one experiment. Automated image acquisition and analysis platforms have been developed as a result. Good examples include Knebel19 and Ritter. The other limitation is on sample work up. During the assay procedure, there are several manipulations that are done to the gel-coated microscope slides. This makes it possible for the gels to become fragile and get damaged in the process thereby risking the experiment. Nevertheless, not using the microscope slides has the potential of increasing the difficulty of performing varied variants like the enzyme-modified ACA associated with the comet assay.

More details about COMPAC-50

We ascertained that the execution of electrophoresis could be done successfully without affecting the size and shape of the comets. For this to happen there is the need to hold the slides in a vertical orientation as well as the convention. In order to achieve similar results to the current ACA, there was no need to increase the electrophoresis duration since it is only the orientation of the slides that had an alteration. The method also allows for the use of multiple slides making it possible for their simultaneous manipulation making it easier for the assay procedure hence requiring minimal skill and it speeds up the process since the slides have the ability of remaining in the racks in all the steps of the comet assay. The aspect also accords the slides protection and works to minimize the gel’s risk of damage.

The HT tank additionally necessitates the use of smaller buffer volumes that is accompanied by cost savings. Running multiple tanks concurrently from a one power supply can help to aid the throughput further. This helps to increase the number of slides run considerably as the bench space requirement increases minimally. This throughput approach has a substantial advantage compared to the existing comet assay procedure and it retains the vital components of the conventional assay at the same time.

Critique

Method 1 can be criticized on the issue of using only 20 comets for ascertaining the statistically significant induction DNA damage. It would have been prudent for the experiment to shed some light on why only 20 comets as it would help interested parties in determining whether this number represented a quality sample size.

Method 2 on the other hand, has been touted to retain the key components of the conventional assay. It would have been good to explain why this is a good thing bearing in mind that the main reason for its enactment is to develop something better than what already exists.

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