Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates.
A high-volume plate-based in situ hybridization 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-fos and 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-fos induction occurred following 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 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-fos mRNA which peaked at 30 min and returned to basal levels within 2 h. Comparison with parallel Northern blotting experiments showed this in situ assay to be at least 20-fold more sensitive and much more rapid to perform.
Harris
D W
DW
Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan 49001, USA.
Kenrick
M K
MK
Pither
R J
RJ
Anson
J G
JG
Jones
D A
DA
D000818:Animals; D016375:Antisense Elements (Genetics); D015152:Blotting, Northern; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D051381:Rats; D012588:Scintillation Counting / Q000295:instrumentation
D016375:Antisense Elements (Genetics); D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases