HIGH VOLUME IN-SITU MRNA HYBRIDIZATION METHOD FOR THE QUANTIFICATION AND DISCOVERY OF DISEASE SPECIFIC GENES

摘要

A high volume substrate-based in-situ hybridization assay has been developed, utilizing scintillant containing microplates. The technique has the sensitivity to reliably detect specific mRNA transcripts at the level of 10-20 copies per cell. High specific activity antisense riboprobes specific for cfos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as a nonhomologous vector derived control probe were used to compare mRNA levels in quiesced rat A-10 smooth muscle cells after stimulation with fetal calf serum (FCS) or platelet-derived growth factor (PDGF). Maximal c-fos induction was shown to occur following stimulation of with 30 ng/ml of PDGF or 10 % FCS, corresponding to a signal from the c-fos probe of 700 cpm. The non-homologous 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 timecourse for c-fos mRNA which peaked at 30 minutes and decreased to less than 50 % of this by 3 hours; a return to background level expression was apparent after 6 hours. Comparison with parallel northern blotting experiment showed this in-situ assay to be at least a 20-fold more sensitive and much more rapid to perform.