Hello everyone,
I have never seen it before but I wonder if that is possible or not? Can I calculate IC50 value from DNA fluorescence? Thanks for your help!
As far as I know, DNA is not itself fluorescent. DNA can be prepared synthetically with fluorescent labels attached to it or with fluorescent nucleotide analogs, which can allow it to be used as part of a fluorescence-based assay for inhibition of enzymes for which DNA is the substrate, such as DNA ligase, DNA polymerase, or topoisomerase. You can also test for DNA binding by compounds by competition with fluorescent DNA binding dyes. Inhibition of fluorescently labeled DNA oligonucleotide binding to proteins can be detected by fluorescence polarization measurements.
I guess the aim of your inclusion of the fluorescent dye to the DNA substrate is to better increase the chances of visibility of the actions & reactions of the nucleotides sequence of the DNA to the target test substrate. Your idea is plausible, therefore, it is very possible to determine or measure the IC50 (In vitro) or ED50 (in vivo) if & only if the fluorescent dye does not interfere with or influence the functions of the DNA substrate
Dear Adam B Shapiro and Peter Etaware thanks for your answers. I wanted to learn if it is possible to use IC50 value determined from the Hoechst DNA fluorescence assay (https://www.ncbi.nlm.nih.gov/pubmed/1489093). I have never encountered a paper used the Hoechst fluorescence method to calculate IC50 or LC50 and I really wonder. I hope I expressed clearly.
I have only read the abstract of the paper, but I don't see any reason why you would not be able to measure a growth IC50 by this method. As I suppose it works, the method is based on there being a direct proportionality between the amount of DNA in the culture, as measured by Hoechst staining, and the number of cells in the culture. This makes sense, as long as you realize that dead cells also contain (or release into the medium) DNA, so this method should be used to measure inhibition of the growth of a culture, rather than cell death.
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