The detection of nucleic acids is important for many life science research tasks, such as sequencing, cDNA synthesis and cloning, RNA transcription and transfection. Accurate detection of DNA/RNA is key to the success of these techniques. DNA/RNA concentration is often measured by UV absorbance at 260 nm. This method, however, is not as accurate and sensitive as DNA quantitation by using fluorescent dyes. AnaSpec, EGT Group is pleased to showcase a variety of premium quality stains, including cell-impermeant and cell-permeant nucleic acid stains.

Cell-impermeant nucleic acid stains

The most popular cell-impermeant nucleic stains are 7-AAD, Ethidium Bromide, Propidium Iodide and Stains-all.

7-AAD (7-aminoactinomycin D) is a fluorescent intercalator that binds selectively to GC regions of DNA. 7-AAD/DNA complexes can be excited by argon-ion laser and emit fluorescence with a maxima of 647 nm, making this nucleic acid stain useful for multicolor fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry.

Ethidium Bromide and Propidium Iodide are classic cell impermeant DNA stains that are structurally similar to phenanthridium intercalators. Both dyes exhibit broad absorption in both UV and visible regions, thus they can be conveniently excited with mercury, xenon-arc lamp or argon-ion laser. This makes them suitable for use in a variety of fluorescence instruments such as fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. EthD-1 and EthD-2 have higher affinity to DNA, RNA and oligonucleotides with a large fluorescence enhancement as compared to ethidium bromide.

Stains-all is a sensitive stain for the electrophoretic characterization of biopolymers on composite agarose-acrylamide gels and hyaluronic acid on polyacrylamide gel. It is suitable for differential staining of nucleic acids and proteins by color: DNA (blue), RNA (bluish purple) and proteins (red).

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Cell-permeant nucleic acid stains

The most popular cell-permeant nucleic acid stains are DAPI and Hoechst dyes, such as Hoechst 33342 and Hoechst 33258.

DAPI is an AT-selective minor groove binder. Binding to dsDNA produces a ~20-fold fluorescence enhancement, emits blue fluorescence upon excitation with mercury-arc lamp or UV lines of the argon-ion laser (Ex/Em = 358/461 nm).

DNA-selective Hoechst dyes are classic minor groove-binding dyes. These common nucleus counterstains bind to DNA, and emit blue fluorescence under light excitation (Ex/Em ~ 350/460 nm). They exhibit relatively large Stokes shifts (~110 nm), and are suitable for multicolor labeling experiments. They also exhibit multiple binding modes and distinct fluorescence spectra that are dependent on dye/base pair ratios.

Dihydroethidium (also known as hydroethidine), the reduced ethidium derivative, is permeant to live cells. It exhibits blue fluorescence in the cytoplasm. After oxidation by many viable cells, the generated ethidium has red fluorescence upon DNA intercalation (Ex/Em = 518/605 nm).

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Examples of cell nuclei staining with nucleic acid stains

Bovine pulmonary artery endothelia cells stained with HiLyte FluorTM 488 and 555. Actins were stained with biotin-conjugated phalloidin, visualized with HiLyte FluorTM 555conjugated streptavidin. Mitochondria were stained with mouse anti-Oxphos complex V primary antibody, visualized with HiLyte FluorTM 488-conjugated goat anti mouse secondary antibody. Nucleus was stained with Hoechst 33342 (Cat# 83218).

Bovine pulmonary artery endothelia cells stained with HiLyte FluorTM 488 and 647. Alpha-tubulins were detected with biotin-conjugated mouse anti-alpha-tubulin antibody, visualized with HiLyte FluorTM 647 conjugated streptavidin. Mitochondria were detected with mouse anti-Oxphos complex V primary antibody, visualized with HiLyte FluorTM 488-conjugated goat anti mouse secondary antibody. Nucleus was stained with DAPI (Cat# 83210).

Alpha-tubulin in bovine pulmonary artery endothelial cells were detected with mouse anti alpha-tubulin primary antibody, visualized with Allophycocyanin (APC)-conjugated goat anti mouse IgG. Nuclei were stained with DAPI (Cat# 83210).

Actins in bovine pulmonary artery endothelial cells were detected with biotin-conjugated phalloidin, visualized with R-phycoerythrin (R-PE)-conjugated streptavidin. Nuclei were stained with Hoechst 33342 (Cat# 83218).