As might be expected, Richard Haugland's posting below on the SYTO dyes is definitive with respect to the SYTO dyes; we may have some disagreements about pyronin (although not about it's not being specific). >There are NO dyes that are SPECIFIC for RNA, including pyronin Y (see abstract >below). > >The SYTO dyes, including the SYTO RED dyes stain both DNA and RNA. Their >quantum yields and affinities may be different but none of the SYTO dyes are >satisfactory for measuring one nucleic acid in the presence of any appreciable >amounts of the other nucleic acid. > >One cannot use the Hoechst and DAPI dyes to "block" DNA and expect a different >dye to then be useful to measure RNA. Binding of these dyes is an equilibrium >and use of excess SYTO Red would eventually displace the Hoechst dyes or DAPI >UNLESS the affinity of that DNA stain were much higher and/or off rate was >very slow, which it is not. I suspect that the SYTO dye may have higher >affinity for DNA than the Hoechst dyes but do not know. > >The Hoechst dyes and DAPI are NOT specific for DNA either. They also bind to >RNA but their quantum yields on RNA are significantly lower and their affinity >for ss nucleic acids is low. > >The abstract (Darzynkiewicz et al, Cytometry 1987 Mar;8(2):138-45; text omitted here) > indicates that it may be possible to combine Hoechst dyes and >pyronin Y because the affinity of pyronin Y for DNA is apparently lower than >that of the Hoechst 33342 dye. The Hoechst/pyronin technique has been around for a while: Shapiro HM: Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry 2:143, 1981 Crissman HA, Darzynkiewicz Z, Tobey RA, Steinkamp JA: Correlated measurements of DNA, RNA and protein in individual cells by flow cytometry. Science 228:1321, 1985 Crissman HA, Darzynkiewicz Z, Tobey RA, Steinkamp JA: Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA and protein content by flow cytometry. J Cell Biol 101:141, 1985 Kapuscinski J, Darzynkiewicz Z: Interactions of pyronin Y(G) with nucleic acids. Cytometry 8:129-136, 1987 Darzynkiewicz Z, Kapuscinski J, Traganos F, Crissman HA: Applications of pyronin Y in cytochemistry of nucleic acids. Cytometry 8:138-145, 1987 Traganos F, Crissman HA, Darzynkiewicz Z: Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells. Exp Cell Res 179:535-44, 1988 Darzynkiewicz et al have shown that pyronin Y bound to single stranded RNA (which is responsible for much of the pyronin absorption when methyl green and pyronin are used in combination for DNA/RNA staining in microscopy and image analysis using transmitted light) is nonfluorescent. The fluorescence comes from pyronin bound to double-stranded RNA, and, where dsRNA either makes up the bulk of or tracks total cellular RNA, the pyronin is usable for quantification of RNA. Srour and his colleagues and other stem cell researchers have used Hoechst 33342/pyronin for vital cell sorting: Ladd AC, Pyatt R, Gothot A, Rice S, McMahel J, Traycoff CM, Srour EF: Orderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0. Blood 1997; 90:658-68. Gothot A, Pyatt R, McMahel J, Rice S, Srour EF: Functional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0 /G1 phase of the cell cycle. Blood 1997; 90:4384-93. Hoechst is not the only dye that can be used with pyronin for DNA and RNA staining; Pollack et al used methyl green to block pyronin staining of DNA: Pollack A, Prudhomme DL, Greenstein DB et al: Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green. Cytometry 3:28, 1982 Toba showed that 7-aminoactinomycin D (7-AAD) could be used in combination with pyronin to measure DNA and RNA content using only 488 nm excitation; Schmid et al have refined this methodology: Toba K, Winton EF, Koike T, Shibata A. Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y. J Immunol Methods 1995;182:193-207. Schmid I, Cole SW, Korin YD, Zack JA, Giorgi JV: Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence. Cytometry, Cytometry 2000 Feb 1;39(2):108-16 I found that oxazine 1, a red-excited tricyclic dye with side chains similar to those of pyronin Y, also produced a "DNA/RNA" like staining pattern when used in combination with Hoechst 33342; however, when the Hoechst/pyronina and Hoechst/oxazine 1 combinations were compared in fixed cells with and without RNAse, much of the oxazine 1 fluorescence was insensitive to RNAse treatment, while 80% or more of the pyronin fluorescence was RNAse-sensitive. Bottom line: I still can't figure out quite why the "DNA specific" dyes combined with pyronin are usable for DNA/RNA content measurement. The DNA content measurements can be quite good in terms of low CV's; the RNA content measurements are obviously less accurate and precise, but probably good enough. From what Dick Haugland has said, it probably isn't worth attempting to substitute SYTO dyes for pyronin in a Hoechst or 7-AAD combination, and we stand united in stating that there are no RNA-specific dyes. In some circumstances, especially if ribosomal RNA makes up a substantial fraction of what you'd want to measure in a cell, it might make sense to use a fluorescently labeled ribosomal RNA probe for quantification; these can even be made specific to species and strains. -Howard
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