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J. Biochem, 1988, Vol. 104, No. 4 595-599
© 1988 Japanese Biochemical Society

research-article

Quantitation of Specific mRNA by RNA-RNA Hybridization Kinetics with Single-Stranded Riboprobes1

Masaaki Tsuda2, Michiko Yagi3, Yushiro Akizawa, Yoshitaka Nagao and Tomofusa Tsuchiya

Faculty of Pharmaceutical Sciences, Okayama University Okayama, Okayama 700

2To whom correspondence should be addressed

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 ß-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of ß-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 ß-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of ß-tubulin mRNA in total brain RNA. By this method, the amounts of ß-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.

1This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

3Present address: Department of Biochemistry, Kawasaki Medical School, Kurashiki, Okayama 701-01.


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