open access

https://hdl.handle.net/2115/43964

Development of an in situ Hybridization Method for Neurohypophysial Hormone mRNAs Using Synthetic Oligonucleotide Probes

英語

1988-04-15

Zoological Society of Japan

Zoological Science

5(2)

397-406

Vasopressin (AVP) and oxytocin (OXT) mRNAS are highly homologous. We developed an in situ hybridization method to discriminate the AVP and the OXT mRNAs using synthetic 22mer deoxyoligonucleotides as probes which have several advantages over the use of cDNAs, e.g., highly specific, easy to obtain a designed probe, and easily accessible to cellular mRNAs. The probes were radiolabeled at the 5' ends with 32P, applied to rehydrated paraffin sections of rat and/or toad hypothalami, and were visualized by autoradiography. RNase treatment before incubation with the probes and measurement of melting temperature showed that the probes actually paired with tissue RNAs. The specificity of hybridization signals was checked by the following tests : absorption test, competition test, a use of alternate probes complementary to the different regions of the same mRNA, cross species hybridization, and comparisons with the immunohistochemical localization of AVP and OXT in adjacent or the same tissue sections. These tests showed that the oligonucleotide probes specifically discriminate the AVP mRNA from the highly homologous OXT mRNA. Furthermore, cross species hybridization clarified that an oligonucleotide probe can discriminate nucleotide sequences which include 2 mismatching bases. The use of multiple probes complementary to different loci in the same mRNA showed not only the specificities of the hybridization signals, but also its usefulness to enhance hybridization signals.

学術雑誌論文

Version of Record

0289-0003