Can anyone suggest a fluorescent DNA-binding dye that gives the highest signal to background ratio in comet assay. Also can anyone suggest a good software that is suitable for yeast comets' analysis.
I agree with Johannes, because ethidium bromide is a unsafe staining. You can obtain SYBR Green easily and you can prepare a diluited stock solution in TE (ph 7.5) which is stable for several weeks when stored at 4 ° C in the dark. SYBR Green’s maximum excitation and emission are respectively 494 nm and 521nm. A fluorescein filter is adequate. The samples fluorescence is usually stable but you can use an antifade solution if fading of samples occurs.
There are several image analysis systems that are suitable for quantitation of Comet Assay data, e.g. 'Comet Assay IV' software but it's a pay-software. In my opinion you can also score your comets by using a free software as Casp: it works very well.
At least 50-75 randomly selected nucleoids should be analyzed per sample.
If you still have any questions, please don't hesitate to contact me
I've been using GelRed in the yeast comet assay. GelRed is a sensitive dye and it only bleaches very slowly (much slower than SYBR Green, from my experience).
We are using EtBr because of it aivailebility and because it is giving very good results (signal-to noise ratio) and does not fade. However, it need to be handled with more care than other stains (like SybrGreen). However, also SybrGreen is an DNA intercalator...
Regarding the software: I rely on Perceptive Instruments Comet Assay IV. It is expensive but give good results in respect to quantitation. Data can be exported and analyzed using statistic tools. Other programs (free: ImageJ plugin, CometScore from TriTek or to be paid: Komet 5.5 Andor Scientific) gave less reliable results.
SYBR Gold stain is >10-fold more sensitive than ethidium bromide for detecting DNA and RNA in denaturing urea, glyoxal, and formaldehyde gels, even with 300 nm transillumination. SYBR Gold stain has also been shown to be much more sensitive than SYBR Green II stain for detecting single strand conformation polymorphism (SSCP) products. Furthermore, it is much more sensitive than silver-staining for the detection of double-stranded DNA in native polyacrylamide gels.
Tuma RS, Beaudet MP, Jin X, Jones LJ, Cheung CY, Yue S, Singer VL. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Anal Biochem (1999) 268:278-288.
I've been using EtBr for many years but NP Singh thinks that YOYO-1 is probably the most appropriate DNA dye (Int J Radiat Bio, 66:23-28, 1994) . However, it is highly probable that this molecule is highly genotoxic!
I would suggest to use 590 nm filter for ethidium bromide. Details are here: http://archive.iorodeo.com/content/comparison-emission-filters-imaging-dna-gels-smartphone
One of the advantages of the assay that has been loosed with the time is that it is very cheap. Propidium iodine is not the best but it is cheap , rapid and effective when you capture 10X fields fast. If you clear the gels with ultrapure water after neutralization and dehydrate with ethanol of high grade the background is low.
I spread 50ul of Propidium iodide (10ug/ml diluted in MilliQ water from 50ug/ml stock) over the slides and cover them (protected from light) during at least 30 min at RT.