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Novel method for the high-throughput processing of slides for the comet assay

View Article: PubMed Central - PubMed

ABSTRACT

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

No MeSH data available.


The effect of comet slide orientation during electrophoresis on comet appearance and quality.HaCaTs were incubated with 100 μM H2O2 prior to analysis by conventional alkaline comet assay, or the new method using the HT rack. Representative images of comets following electrophoresis performed in the same electrophoresis tank with the comet slides held (A) vertically in a HT rack, and (B) horizontally, as is the convention. (C) Quantification of H2O2-induced DNA damage in HaCaTs determined by ACA with electrophoresis performed in either the horizontal or vertical orientation. Error bars represent the median and max/min of 200 individual determinations from two independent experiments (ns = not significant).
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f3: The effect of comet slide orientation during electrophoresis on comet appearance and quality.HaCaTs were incubated with 100 μM H2O2 prior to analysis by conventional alkaline comet assay, or the new method using the HT rack. Representative images of comets following electrophoresis performed in the same electrophoresis tank with the comet slides held (A) vertically in a HT rack, and (B) horizontally, as is the convention. (C) Quantification of H2O2-induced DNA damage in HaCaTs determined by ACA with electrophoresis performed in either the horizontal or vertical orientation. Error bars represent the median and max/min of 200 individual determinations from two independent experiments (ns = not significant).

Mentions: 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 with performing ACA in the conventional, horizontal orientation. The results showed that the orientation and the shape of the comets which were run vertically in the HT rack (Figure 3A) were identical to those run horizontally (Figure 3B). Furthermore the data obtained after scoring the comets indicated that there was no significant difference in percentage tail DNA between the samples run horizontally or vertically (P > 0.05; Figure 3C). Additionally, using the HT racks provided a 60% decrease in time spent manipulating slides (i.e. Figure 1, steps III, IV, V, VI, VII, VIII, IX, X, XI, XII), compared to conventional ACA, together with decreasing the risk of damage to gels during manipulation.


Novel method for the high-throughput processing of slides for the comet assay
The effect of comet slide orientation during electrophoresis on comet appearance and quality.HaCaTs were incubated with 100 μM H2O2 prior to analysis by conventional alkaline comet assay, or the new method using the HT rack. Representative images of comets following electrophoresis performed in the same electrophoresis tank with the comet slides held (A) vertically in a HT rack, and (B) horizontally, as is the convention. (C) Quantification of H2O2-induced DNA damage in HaCaTs determined by ACA with electrophoresis performed in either the horizontal or vertical orientation. Error bars represent the median and max/min of 200 individual determinations from two independent experiments (ns = not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384090&req=5

f3: The effect of comet slide orientation during electrophoresis on comet appearance and quality.HaCaTs were incubated with 100 μM H2O2 prior to analysis by conventional alkaline comet assay, or the new method using the HT rack. Representative images of comets following electrophoresis performed in the same electrophoresis tank with the comet slides held (A) vertically in a HT rack, and (B) horizontally, as is the convention. (C) Quantification of H2O2-induced DNA damage in HaCaTs determined by ACA with electrophoresis performed in either the horizontal or vertical orientation. Error bars represent the median and max/min of 200 individual determinations from two independent experiments (ns = not significant).
Mentions: 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 with performing ACA in the conventional, horizontal orientation. The results showed that the orientation and the shape of the comets which were run vertically in the HT rack (Figure 3A) were identical to those run horizontally (Figure 3B). Furthermore the data obtained after scoring the comets indicated that there was no significant difference in percentage tail DNA between the samples run horizontally or vertically (P > 0.05; Figure 3C). Additionally, using the HT racks provided a 60% decrease in time spent manipulating slides (i.e. Figure 1, steps III, IV, V, VI, VII, VIII, IX, X, XI, XII), compared to conventional ACA, together with decreasing the risk of damage to gels during manipulation.

View Article: PubMed Central - PubMed

ABSTRACT

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

No MeSH data available.