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 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.
Cytometry 1987 Mar;8(2):138-45
Application of pyronin Y(G) in cytochemistry of nucleic
acids.
Darzynkiewicz Z, Kapuscinski J, Traganos F, Crissman
HA.
Chinese hamster ovary (CHO) cells or isolated nuclei
were stained with pyronin Y(PY) and analyzed by absorption or
fluorescence microscopy, as well as by flow cytometry.
Specificity of the staining reaction was assayed by testing sensitivity of
the stainable material to RNase or DNase. The colored
complexes detected by light absorption in fixed cells stained with PY are
nonfluorescent and are most likely the products of
condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG)
are the most sensitive to condensation. The products of
PY interaction with double-stranded (ds) nucleic acids are fluorescent
and can be detected in cells by cytofluorometry. PY
used alone stains both DNA and RNA, and the staining capabilities of these
nucleic acids vary depending upon the PY concentration
at equilibrium; at a concentration above 330 microM, the RNA
stainability decreases, perhaps due to its denaturation
and condensation caused by the dye. In the presence of Hoechst 33342,
PY can specifically stain RNA in fixed cells or
isolated cell nuclei. Because only complexes of PY with ds RNA are
fluorescent, this
dye can be used as a probe of RNA conformation, e.g.,
to monitor denaturation of RNA in situ. The RNA stainability of mitotic
cells is about 25% lower than that of cells in G2
phase, which indicates that during mitosis proportionately less cellular RNA
is in
the ds conformation. The advantages and limitations of
the two cytochemical methods for DNA/RNA detection, one based on the
use of Hoechst 33342 and PY, and another employing the
metachromatic properties of acridine orange, are compared.
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?
>
> Thanks for your attention!
>
> Elena.
>
> -----------------------------------------
> Elena Soriano
> c/o Institut für Angewandte Mikrobiologie
> Univ.f.Bodenkultur Wien
>
> Tel: +43 1 36006 6241
> Fax: +43 1 36 97 615
> http://www.boku.ac.at/iam
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