Gene expression profiling of HIV-1 infection using cDNA microarrays.
To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.
van 't Wout
Angélique B
AB
D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism / Q000821:virology; D002460:Cell Line; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods