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Microarray Data Link
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 http://www.grc.nia.nih.gov/branches/rrb/dna/array.htm.
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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