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

Abstract

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.

Androgenic Regulation of Expression of β‐Tubulin Messenger Ribonucleic Acid in Motoneurons of the Spinal Nucleus of the Bulbocavernosus

“…Northern blot analysis revealed a band of P-tubulin mRNA of about 1,800 bases ( Fig. 2) (33,34). These results confirm the specificity of the cDNA probe.…”