title: Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses.
abstract: 8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells.
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
authors: Kaley M
authors: KM
authors: Wilburn
authors: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi.|Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK.
authors: Henry C
authors: HC
authors: Mwandumba
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.|Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi.|Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK.
authors: Kondwani C
authors: KC
authors: Jambo
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
authors: Saikat
authors: S
authors: Boliar
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
authors: Sabrina
authors: S
authors: Solouki
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
authors: David G
authors: DG
authors: Russell
authors: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA. dwg62@cornell.edu.
authors: David W
authors: 0000-0002-3537-4585
authors: DW
authors: Gludish
mesh_terms: D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D005434:Flow Cytometry; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D011533:Proviruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D014157:Transcription Factors
keywords: 8E5; FISH:FACS; FISH:FLOW; HIV RNA in situ hybridization; HIV cell-associated RNA; HIV flow cytometry assay; HIV proviral loss; HIV proviral silencing; Reservoir quantitation