NONRADIOACTIVE QUANTIFICATION OF GLUCOCORTICOID RECEPTOR EXPRESSION DURING DIFFERENTIATION OF HUMAN MONOCYTIC CELLS (U937)

We describe a method for relative quantification of specific mRNA usin g a nonradioactive assay based on DNA strand competition between ident ical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quant ity increased, that of the double-labeled probe decreased in accordanc e with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 ce lls before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-tr ans retinoic acid (RA) and 1,25-dihydroxyvitamin D-3 (VD). We observed that TPA treatment was associated with an increase in specific bindin g of [H-3]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment. (C) 1995 Academic P ress, Inc.