Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes.
A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.
Tsuda
M
M
Faculty of Pharmaceutical Sciences, Okayama University.
Yagi
M
M
Akizawa
Y
Y
Nagao
Y
Y
Tsuchiya
T
T
D000818:Animals; D001921:Brain / Q000254:growth & development; D001923:Brain Chemistry; D003001:Cloning, Molecular; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics*
D015347:RNA Probes; D012333:RNA, Messenger; D014404:Tubulin