A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA

R. M. Borzì, A. Piacentini, M. C G Monaco, G. Lisignoli, A. Degrassi, L. Cattini, S. Santi, A. Facchini

Research output: Contribution to journalArticle

Abstract

In HIV patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-1. The cells were fixed in suspension and hybridized by means of an HIV-1 genomic probe labeled with digoxigenin-11-dUTP. An FITC-labeled anti-diooxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Flow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples.

Original languageEnglish
Pages (from-to)167-176
Number of pages10
JournalJournal of Immunological Methods
Volume193
Issue number2
DOIs
Publication statusPublished - Jun 21 1996

Keywords

  • Flow cytometry
  • Fluorescent in situ hybridization
  • HIV-RNA

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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