Manitoba HIV Research Group University of Manitoba





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MIAME Information Sheet

Sample Collection and Processing

Fifty ml of blood was collected from an HIV infected individual and an age and sex-matched uninfected control in tubes containing heparin at the St. Bonniface Hospital Research Centre and the University of Manitoba, respectively. Peripheral blood mononuclear cells were isolated from whole blood by ficoll density gradient centrifugation using standard methods. Isolated cells were counted and tested for viability by Trypan blue exclusion prior to culture.

Cell Culture and T-lymphocyte Subset Purification

To allow normal antigenic processing, presentation and cell type interaction, we performed all stimulations in whole PBMC population. PBMC were incubated at 2.0 x 106 cells per ml in RPMI/10% FCS + 2% pen/strep. PBMC were stimulated with either the fungus Candida albicans (Grier Laboratories) at 10ug/ml, recombinant HIV p24 protein produced in our lab @ 1ug/ml or left unstimulated for 24 hours at 370C + 5% CO2. Following stimulation, cells were collected, washed twice in PBS + 2% FCS and split into 3 groups for sub-population purification. Highly purified populations of CD4+ or CD8+ T-lymphocytes were obtained using a magnetic bead negative selection procedure (StemCell Technologies, Vancouver, BC) according to the instructions of the manufacturers. Briefly, PBMC were bound to a mixture of antibodies for negative selection of either CD4+ or CD8+ T-lymphocytes followed by binding of a magnetic bead coupled secondary antibody. Cells were passed over columns in the presence of magnetic field for T-lymphocyte purification. Negative selection was performed to limit cellular activation from the separation antibodies. In our hands, purified cell populations have consistently been shown to be >95% pure by flow cytometry (data not shown).

RNA Isolation and Quantification

Total cellular RNA was isolated from at least 106 PBMC, CD4+ and CD8+ T-lymphocytes from each study individual for all stimulation conditions (media alone, C. albicans, p24) using RNeasy mini kits (Qiagen) according to manufacturers instructions. RNA quantity was measured by UV spectrometry and for quality by amplification of message RNA by glyceraldehydes phosphate dehydrogenase RT-PCR (data not shown).

cDNA Microarrays

Immune microarrays were obtained from the National Institute on aging. Information on the array used (human immune focused 4,608 gene set) is available at RNA labeling and array hybridization was carried out as described (5, 26). Briefly, one microgram of total cellular RNA from each sample was reverse transcribed using oligo dT primers and labeled with 33P-dCTP (NEN) using LabelStar Array kits (Qiagen) according to manufacturers instructions. Labeled cDNA was hybridized to nylon human immune arrays (4,608 genes), obtained through collaboration from the National Institute on Aging (20) in 5ml Microhyb (ResGen) buffer in the presence of PolyA (Sigma) and human cotI DNA (Invitrogen) at 42OC for 18 hours. Arrays were washed in 2X SSC + 1% SDS twice for 15 minutes and exposed to phosphorimage screens for at least 24 hours. Images were obtained using the BioRad personal Fx phosphorimager and Quantity One software (BioRad). Quantification of spot values was carried out using ArrayPro software.Quantified data files were exported to Microsoft Excel to undergo averaging of duplicate spots and pre-filtering. Briefly, all duplicate spots with values having a variance of greater than 0.2 were eliminated from further analysis. Data was transferred to GeneSpring (Silicon Genetics) for data normalization and comparison. All data was subjected to at least one per-chip and one per-gene normalization step in order to control for variation between different arrays. Genes were considered to be significantly changed in expression if normalized values from the stimulated condition (C. albicans/p24) were 2 fold greater than or less than the unstimulated (media alone) condition. Gene lists of significant changers (both up and down-regulated) were created for each patient for each cell type and stimulation condition and compared by Venn diagram using GeneSpring software. Quantified raw data files for all arrays are available at this site.

Candida Simulation

Media Simulation

P24 Simulation

Nylon Array Information

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Page last modified: 31/08/2009