A FLUORESCENT IN-SITU HYBRIDIZATION METHOD IN FLOW-CYTOMETRY TO DETECT HIV-1 SPECIFIC RNA

In HIV+ patients, the presence of HIV-RNA in plasma and circulating ce lls has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have d eveloped a reliable fluorescent in situ hybridization method for the d etection of HIV specific RNA by flow cytometry. The procedure was appl ied to a panel of chronically infected cell lines and to an acutely in fected cell line mimicking normal peripheral blood lymphocytes in susc eptibility 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 FTTC-labeled anti-digoxigenin antiserum was then applied and the re sulting 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 deve loped. Flow cytometric detection of in situ hybridization offers the p ossibility of analyzing thousands of cells in a few seconds and of col lecting multiparametric information at the single cell level, thus pro viding a potential tool for detecting the rare HIV-RNA expressing cell s in peripheral blood samples.