RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization

We describe the development of a sensitive fluorescence-based solution assa y for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen re agent exhibits >1000-fold fluorescence enhancement and high quantum yield ( 0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a l arge extinction coefficient (67,000 cm(-1) M-1). The RiboGreen assay allows detection of as little as 1.0 ng/ml RNA in a standard fluorometer, filter fluorometer, or fluorescence microplate reader-surpassing the sensitivity a chieved with ethidium bromide by 200-fold, The linear quantitation range fo r RiboGreen reagent extends over three orders of magnitude in RNA concentra tion. Using 750 nM RiboGreen reagent, we quantitated 20 ng/ml to 1.0 mu g/m l RNA. By diluting the reagent to 75 nM, we could quantitate 1.0 to 50 ng/m l RNA. Both assay ranges exhibited linear fluorescence increases versus RNA concentration (r(2) = 0.999). Assay linearity was maintained in the presen ce of salts, protein, urea, ethanol, chloroform, agarose, and some detergen ts. Several different RNA types yielded similar signal intensities and dete ction sensitivities. The assay is easy to use, rapid, and readily adaptable for automation. (C) 1998 Academic Press.