A solution hybridization method for quantification of mRNAs: determining the amount and stability of oncogene mRNA.
A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.
Tenhunen
J
J
Orion Corporation, Orion Pharmaceutica, Biotechnology, Helsinki, Finland.
Eloranta
J
J
Kallio
A
A
Söderlund
H
H
D015342:DNA Probes; D016264:Dextran Sulfate / Q000494:pharmacology; D005559:Formamides / Q000494:pharmacology; D016259:Genes, myc; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D012996:Solutions; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D005559:Formamides; D015347:RNA Probes; D012333:RNA, Messenger; D012996:Solutions; C031066:formamide; D016264:Dextran Sulfate