Development of a High-Volumein SitumRNA Hybridization Assay for the Quantification of Gene Expression Utilizing Scintillating Microplates

A high-volume plate-basedin situhybridization assay has been developed, utilizing Amersham Cytostar-T scintillating microplates. This assay reliably detects specific mRNA transcripts at the level of 10–20 copies per cell. Radiolabeled antisense riboprobes specific for c-fosand glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as nonhomologous 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-fosinduction occurred following stimulation of A10 cells with 30 ng/ml of PDGF, corresponding to a signal from the c-fosprobe of 700 cpm. The nonhomologous control background 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 cells were stimulated at various times to provide an induction time course for c-fosmRNA which peaked at 30 min and returned to basal levels within 2 h. Comparison with parallel Northern blotting experiments showed thisin situassay to be at least 20-fold more sensitive and much more rapid to perform.