DEVELOPMENT OF A HIGH-VOLUME IN-SITU MESSENGER-RNA HYBRIDIZATION ASSAY FOR THE QUANTIFICATION OF GENE-EXPRESSION UTILIZING SCINTILLATING MICROPLATES

A high-volume plate-based in situ hybridization assay has been develop ed, utilizing Amersham Cytostar-T scintillating microplates. This assa y reliably detects specific mRNA transcripts at the level of 10-20 cop ies per cell. Radiolabeled antisense riboprobes specific for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as nonhomolo gous vector-derived control probes were used to compare mRNA levels in quiesced rat A10 smooth muscle cells after stimulation with platelet- derived growth factor (PDGF). Maximal c-fos induction occurred followi ng stimulation of A10 cells with 30 ng/ml of PDGF, corresponding to a signal from the c-fos probe of 700 cpm. The nonhomologous control back ground of 50 cpm and the GAPDH signals of 1700 cpm were independent of stimulation with PDGF or serum. Using PDGF, at 30 ng/ml, quiesced cel ls were stimulated at various times to provide an induction time cours e for c-fos mRNA which peaked at 30 min and returned to basal levels w ithin 2 h. Comparison with parallel Northern blotting experiments show ed this in situ assay to be at least 80-fold more sensitive and much m ore rapid to perform. (C) 1996 Academic Press, Inc.