Thiazole Orange

Thiazole Orange is an asymmetric anthocyanin dye that can be coupled with oligonucleotides (ONs) to prepare fluorescent hybridization probes. Thiazole Orange has been widely used in biomolecular detection and staining of DNA/ RNA in gels and can be used for reticulocyte analysis. Thiazole orange generates a significant fluorescence enhancement and high quantum yield when it binds with nucleic acids, especially RNA. Thiazole orange can permeate living cell membranes. Thiazole orange can use UV light for detection, but can also be detected with blue light. The excitation and emission of Thiazole orange are λ_ex = 510 nm (488 nm and 470 nm also show strong excitation) and λ_em = 527 nm, respectively^{[1][2][3][4][5][6]}. In Vitro:Preparation of Thiazole Orange Staining Solution:
1.1 Preparing the Storage Solution
Prepare a 1 mg/mL solution of Thiazole Orange using methanol. For example, dissolve 1 mg of Thiazole Orange in 1 mL of methanol.
1.2 Preparing the working solution.
Dilute the 1 mg/mL Thiazole Orange stock solution in phosphate buffered saline containing 0.002 M EDTA and 0.02% sodium azide at a ratio of 1:10000.
Thiazole Orange staining
1 Reticulocyte analysis^[1]
1)Whole blood (5 pL) is added to the diluted dye solution (1 mL) in a 12 x 75 mm culture tube and incubated at room temperature for 1 h. The sample is analyzed on a FACS IV, FACS 440, or FACStar.
2)Data analysis (flow cytometry) : Mature red blood cells between the left and middle marker and reticulocytes between the middle and right marker. The classification of cell type is determined through the analysis of mixed whole blood from human peripheral mononuclear cells.
2 DNA Electrophoresis^[3]
1)Mix agarose (~1% w/v, percentage can be varied for particular size separations) in buffer (approximately 70 mL for a mini-gel (8 x 7 cm)). Buffers are commonly TAE (tris-acetate-EDTA, 40 mM Tris, 20 mM acetate, 1 mM EDTA, pH approximately 8.6) or TBE (tris-borate-EDTA, 90 mM Tris, 90 mM borate, 2 mM EDTA, pH approximately 8.3)).
2)Add thiazole orange to a final concentration of 1.3 μg/mL.
NOTE: The gel can also be stained with Thiazole Orange after electrophoresis.
3)Microwave the mixture of agarose, buffer, and thiazole orange to dissolve agarose (approximately 60 s). This step is commonly referred to as “melting”. Swirl (5 s) to aid dissolution if needed. Allow the agarose solution to solidify into a gel.
4)Place the gel in the electrophoresis apparatus. Load DNA samples (commonly 10 μL) using a loading dye. Attach the cover and electrodes. Apply voltage (typically ~100V for a mini-gel) until loading dye has traveled an appropriate distance (approximately 4-7 cm for a mini-gel).
NOTE: Thiazole orange is positively charged, migrating in the opposite direction of electrophoresing DNA.
5)Remove the gel from the electrophoresis apparatus and place on a UV transilluminator and a blue-light transilluminator (~470 nm maximum emission wavelength).
Select appropriate excitation and emission settings in gel-imaging apparatus. The excitation and emission of thiazole orange (λ_ex,max = 510 nm (488 nm and 470 nm also show strong excitation, in addition to strong excitation at UV wavelengths); λ_em = 527 nm).