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 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-digoxigenin 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.
Borzì
R M
RM
Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, I.O.R., Bologna, Italy.
Piacentini
A
A
Monaco
M C
MC
Lisignoli
G
G
Degrassi
A
A
Cattini
L
L
Santi
S
S
Facchini
A
A
D015703:Antigens, CD / Q000032:analysis / Q000235:genetics; D002460:Cell Line; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D016655:HIV Core Protein p24 / Q000032:analysis / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*
D015703:Antigens, CD; D005404:Fixatives; D016655:HIV Core Protein p24; D012367:RNA, Viral