Elena Soriano wrote- >My intention is to use SYTO red for RNA specific staining like I use >Pyronine- Y. Does anyone knows if this possible? > >When I stain the DNA with Hoechst or DAPI (selective for DNA), the >Pyronine-Y finds no place in these molecules, and therefore binds only to >the RNA, wich is then the only nucleic acid that has free binding points. > >-> Could I block the DNA with Hoechst or DAPI and then stain with SYTO red >to detect the RNA? > >Would SYTO red still find free binding points when the DNA is allready >stained with one of these DNA selective fluorochromes? This is an interesting question. Pyronin Y is believed to stain double-stranded (e.g., ribosomal) RNA, and its structural similarity to acridine orange suggests an intercalative binding mechanism for the staining. As Ingrid Schmid et al have shown, pyronin Y also stains DNA; again, one would presume binding to be intercalative. However, the DNA-specific dyes which demonstrably block pyronin Y staining of DNA (the list includes the Hoechst dyes and DAPI, which are fluorescent, and methyl green, which is not, at least when excited with blue-green or green light) are outer groove binders which bind to to A-T triplets. I haven't seen anything on the molecular mechanism of the blocking; one possibility is that the outer groove binding of the DNA-specific stain unwinds the double helix somewhat, preventing intercalative binding of tricyclic dyes such as pyronin Y. The SYTO dyes are said to bind intercalatively to both double-stranded DNA and double-stranded RNA; their fluorescence enhancement on binding is substantially larger than the fluorescence enhancement of Hoechst dyes or DAPI (and even higher than pyronin fluorescence enhancement, which is minimal). Whether the DNA-specific dyes would block binding and fluorescence of SYTO dyes is not clear a priori; you'd have to do the experiment. A good test subject would be a stimulated lymphocyte culture; the idea would be to compare 2-dimensional displays of Hoechst/pyronin- and Hoechst/SYTO red-stained cells. Lymphoblasts in culture typically don't have the G0 (or G1q) subpopulation, but you could compare them to peripheral blood lymphocytes, the great majority of which are normally in G1q. A few years back, we tried several styryl and cyanine dyes which stain both DNA and RNA in combination with Hoechst dyes; we did not find the blocking effect (unpublished). The cyanine dyes we looked at were probably similar to SYTO red in structure, but it's still worthwhile doing the experiment. In theory, a mixture of two dyes which stain both DNA and RNA, but with different affinities (one more DNA-specific, the other more RNA-specific), could be used to determine DNA and RNA content using the same mathematics used for immunofluorescence compensation; I don't think anybody has actually done this yet. If I get around to playing with any of this in the next few months, I'll probably put it in the 4th Edition of Practical Flow Cytometry. -Howard
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