发明名称 |
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 c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as a non-homologous 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 time-course 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. |
申请公布号 |
AU2542497(A) |
申请公布日期 |
1997.11.12 |
申请号 |
AU19970025424 |
申请日期 |
1997.04.03 |
申请人 |
PHARMACIA & UPJOHN COMPANY;AMERSHAM INTERNATIONAL PLC |
发明人 |
DAVID A. JONES;DOUGLAS W HARRIS;MICHAEL K. KENRICK;NICHOLAS THOMAS |
分类号 |
G01N33/53;C12N15/09;C12Q1/68;G01N33/566 |
主分类号 |
G01N33/53 |
代理机构 |
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代理人 |
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主权项 |
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地址 |
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