title: Detection of messenger RNA and low-abundance heteronuclear RNA with single-stranded DNA probes produced by amplified primer extension labeling.
abstract: We describe a procedure for detection of low-abundance cellular RNAs by in situ hybridization histochemistry, using single-stranded DNA probes produced by amplified primer extension labeling with Taq polymerase. We have used this approach to detect a number of high- and low-abundance RNA species and have found it to be a simple and reproducible method of obtaining sensitive probes for in situ hybridization studies. For example, DNA probes generated by amplified primer extension labeling can detect low-abundance heteronuclear RNAs in individual neurons. Since this procedure does not involve recombinant DNA technology or microbiological facilities, it should prove useful to a wide variety of investigators studying the regulation of gene expression at the cellular level.
authors.affiliation: Laboratory of Neurobiology and Behavior, Rockefeller University, New York, New York.
authors.forename: P J
authors.initials: PJ
authors.lastname: Brooks
authors.forename: M G
authors.initials: MG
authors.lastname: Kaplitt
authors.forename: S P
authors.initials: SP
authors.lastname: Kleopoulos
authors.forename: T
authors.initials: T
authors.lastname: Funabashi
authors.forename: C V
authors.initials: CV
authors.lastname: Mobbs
authors.forename: D W
authors.initials: DW
authors.lastname: Pfaff
mesh_terms: D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D012332:RNA, Heterogeneous Nuclear / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D019914:Taq Polymerase
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase