title: DoubleIn situHybridization for MicroRNAs and mRNAs in Brain Tissues.
abstract: MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding thein vivolocalization of miRNAs is an important step for uncovering their roles in brain function. However, thein situdetection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization ofin situhybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37°C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 × standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqManTMprimers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in thein vivolocalization of miRNAs, and the elucidation of the specific roles of miRNAs.
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kasai
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Sora
authors.initials: S
authors.lastname: Kakihara
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Miura
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Okada
authors.affiliation: Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui Suita, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hayata-Takano
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Hazama
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Misaki
authors.initials: M
authors.lastname: Niu
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University Suita, Japan.
authors.forename: Norihito
authors.initials: N
authors.lastname: Shintani
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka UniversitySuita, Japan; iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka UniversitySuita, Japan; Department of Pharmacology, Graduate School of Dentistry, Osaka UniversitySuita, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Nakazawa
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka UniversitySuita, Japan; Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of FukuiSuita, Japan; iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka UniversitySuita, Japan; Division of Bioscience, Institute for Datability Science, Osaka UniversitySuita, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hashimoto
keywords: in situ hybridization; miR-100; miR-181a; miR-219; miR-34a; miR-9; miRNA; parvalbumin