Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937).
We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment.
Roux
S
S
Centre de Recherche INSERM, Montpellier, France.
Térouanne
B
B
Defacque
H
H
Vachier
I
I
Loubatière
J
J
Nicolas
J C
JC
D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D003907:Dexamethasone / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone