Laurie J. Jones; Stephen T. Yue; Ching-Ying Cheung; Victoria L. Singer (1998). RNA Quantitation by Fluorescence-Based Solution Assay: RiboGreen Reagent Characterization. , 265(2), 368–374. doi:10.1006/abio.1998.2914
RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits >1000-fold fluorescence enhancementand high quantum yield (0.65) upon binding nucleicacids, with excitation and emission maxima near thoseof fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coefficient (67,000 cm21M21). The RiboGreen assay allows detection of as littleas 1.0 ng/ml RNA in a standard fluorometer, filter fluorometer, or fluorescence microplate reader—surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range forRiboGreen reagent extends over three orders of magnitude in RNA concentration. Using 750 nM RiboGreen reagent, we quantitated 20 ng/ml to 1.0 mg/mlRNA. By diluting the reagent to 75 nM, we could quantitate 1.0 to 50 ng/ml RNA. Both assay ranges exhibitedlinear fluorescence increases versus RNA concentration (r2 5 0.999). Assay linearity was maintained in thepresence of salts, protein, urea, ethanol, chloroform,agarose, and some detergents. Several different RNAtypes yielded similar signal intensities and detectionsensitivities. The assay is easy to use, rapid, andreadily adaptable for automation
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