title: In situ hybridization AT-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalin-fixed and paraffin-embedded tissues.
abstract: In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakajima
authors.forename: Petronela
authors.initials: P
authors.lastname: Ionescu
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.forename: Michie
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kuroita
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshikura
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Sata
mesh_terms: D000225:Adenine; D000328:Adult; D000956:Antigens, Viral / Q000032:analysis; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D015342:DNA Probes; D005260:Female; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007223:Infant; D008198:Lymph Nodes / Q000473:pathology* / Q000821:virology; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013941:Thymine
chemical_list: D000956:Antigens, Viral; D015342:DNA Probes; D012333:RNA, Messenger; D000225:Adenine; D013941:Thymine