Identification of differentially expressed mRNA species during HIV infection by RNA arbitrarily primed PCR.
BACKGROUND
A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression.


OBJECTIVE
To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR.


METHODS
RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets.


RESULTS
Three genes, gamma-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of gamma-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that gamma-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation.


CONCLUSIONS
These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.
Boyer
V
V
Immune Response Corporation, Carlsbad, CA, USA. boyer@lyonl51.inserm.fr
Pezzoli
P
P
Audoly
G
G
Desgranges
C
C
Jensen
F
F
Ferre
F
F
D000199:Actins / Q000378:metabolism; D001483:Base Sequence; D016172:DNA Fingerprinting; D004279:DNA, Viral; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D000199:Actins; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral