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

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Title: Development of an in situ Hybridization Method for Neurohypophysial Hormone mRNAs Using Synthetic Oligonucleotide Probes
Authors: Hyodo, Susumu Browse this author
Fujiwara, Mamoru Browse this author
Kozono, Shigeru Browse this author
Sato, Moriyuki Browse this author
Urano, Akihisa Browse this author
Issue Date: 15-Apr-1988
Publisher: Zoological Society of Japan
Journal Title: Zoological Science
Volume: 5
Issue: 2
Start Page: 397
End Page: 406
Abstract: 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.
Type: article
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 浦野 明央

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