Detection of human T lymphotrophic virus type I (HTLV-I) DNA and mRNA in individual cells by polymerase chain reaction (PCR) in situ hybridization (ISH) and reverse transcription (RT)-PCR ISH.
Human T lymphotrophic virus type I (HTLV-I) proviral DNA and mRNA in the blood obtained directly from HTLV-I infected adult T cell leukemia (ATL) patients were amplified by the polymerase chain reaction (PCR), and reverse transcription (RT)-PCR, and then were hybridized to fluorescein-labelled probes by means of in situ hybridization (ISH). Before the cytospin samples were prepared, heterogenous cell populations were reproducibly resolved into HTLV-I-positive and -negative distributions. Immunohistochemical staining was performed, using anti-fluorescein monoclonal antibody. Microscopic observations demonstrated a preserved cellular morphology. The intranuclear localization of amplified DNA products of proviral HTLV-I by PCR/ISH, and intracytoplasmic localization of amplified DNA of HTLV-I tax/rex mRNA by RT-PCR/ISH were maintained. In this study, about one in 10 HTLV-I provirus integrated cells expressed low copies of tax/rex mRNA. In HTLV-I-negative cell lines, amplified DNA was not observed by either PCR/ISH or RT-PCR/ISH. With the use of this technique it is thus possible to detect single-copy DNA and a few copies of mRNA, and it is therefore possible to study, not only suspended materials, but also other tissue materials for further characterization, in association with the localization of the HTLV-I infected cells.
Ohshima
K
K
Department of Internal Medicine, Kagoshima University, Japan.
Hashimoto
K
K
Izumo
S
S
Suzumiya
J
J
Kikuchi
M
M
D004279:DNA, Viral / Q000032:analysis*; D018922:HL-60 Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D015458:Leukemia, T-Cell / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis*
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral