Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Gene expression profiling of microglia infected by a highly neurovirulent murine leukemia virus: implications for neuropathogenesis

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We undertook a survey of gene expression changes in primary microglial cultures with and without neurovirulent (FrCasE) and non-neurovirulent (Fr57E) virus infection to identify physiological changes that could be relevant to the induction of spongiform neurodegeneration. These gene expression analyses were performed using Affymetrix 430A mouse GeneChips (5 chips for each of the three experimental conditions, representing over 14,000 murine genes and ESTs. RNA from 5 separate microglial culture preparations were analyzed for Control (mock infected), Fr57E-, and FrCasE-infected microglia. Present/absent calls were based on MicroArray Suite 5.0 from Affymetrix. Affymetrix CEL files were analyzed using dChip software after normalization of the data between all 15 arrays. Statistical analyses were performed using ANOVA.

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Transcription profiling by array of mouse primary microglial cells infected with neurovirulent (FrCasE) and non-neurovirulent (Fr57E) virus

Transcription profiling of mouse primary microglial cultures with and without neurovirulent (FrCasE) and non-neurovirulent (Fr57E) virus infection

We undertook a survey of gene expression changes in primary microglial cultures with and without neurovirulent (FrCasE) and non-neurovirulent (Fr57E) virus infection to identify physiological changes that could be relevant to the induction of spongiform neurodegeneration. These gene expression analyses were performed using Affymetrix 430A mouse GeneChips (5 chips for each of the three experimental conditions, representing over 14,000 murine genes and ESTs. RNA from 5 separate microglial culture preparations were analyzed for Control (mock infected), Fr57E-, and FrCasE-infected microglia. Present/absent calls were based on MicroArray Suite 5.0 from Affymetrix. Affymetrix CEL files were analyzed using dChip software after normalization of the data between all 15 arrays. Statistical analyses were performed using ANOVA.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Microglial cultures were prepared from neonatal (P0) IRW mouse brains from which the cerebellum, olfactory bulbs, and meninges had been removed as previously described. Briefly, brains were triturated, passed through a 70 µm nylon mesh filter, treated for 5 minutes at 37C with 0.25% Trypsin / 1 mM EDTA, and then quenched by the addition of newborn calf serum (Hyclone) to 2%. Mixed CNS cells were plated in T75 Corning flasks (approximately 1 brain equivalent per flask) in Advanced D-MEM (Gibco) supplemented with 5% fetal bovine serum, 4 mM glutamine, penicillin and streptomycin (DMA5). After 7-10 days in culture, when the mixed glia reached approximately 50% confluence, the cultures were infected with FrCasE or Fr57E MLVs or mock infected at a MOI of ~1 in the presence of 8 µg/ ml of polybrene. After 1 hour exposure to virus/polybrene, medium was changed to DMA5 supplemented with 0.5 ng/ml GM-CSF to enhance the proliferation of microglia.  When abundant phase bright cells could be observed over a confluent mixed glial monolayer, microglia were isolated by shake off, plated on fresh 10-cm Corning tissue culture plates for 1 hour, followed by 3X PBS washes to remove non-adherent cells. Parallel cultures of adherent cells were routinely checked for purity using antibodies directed against F4/80, and CD11b, by indirect immunofluorescence. The presence of virus infection was determined by staining microglia with monoclonal antibodies that were specific to either CasBrE or Friend Env. All microglial cultures tested were determined to be greater than 98% pure with essentially 100% infection by the appropriate virus.

Mixed glia were grown in Advanced D-MEM (Gibco) supplemented with 5% fetal bovine serum (Hyclone), glutamine, and antibiotics (DMA5) until cells reached approximately 50% confluence (7-10 days in culture) at which time they were infected with the highly neurovirulent ecotropic murine leukemia virus FrCasE at a multiplicity of infection of ~1 infectious unit per cell in the presence of 8 µg/ ml of polybrene. After 1 hour of virus/polybrene exposure, medium was replaced with fresh DMA5 containing 0.5 ng/ml GM-CSF. Cultures were grown until they were confluent and abundant phase bright cells appeared growing above the monolayer. Microglia were isolated by shake off, plated for 1 hour on Corning 10-cm tissue culture plates, washed 3 times with PBS, and then extracted with TRIzol for RNA isolation according to the manufacturer's instructions.

Mixed glial cultures were generated from a pool of 8-10 P0 neonatal brains. Approximately 1 brain equivalent of this pool was seeded in each tissue culture flask to generate each mixed glial culture. Each flask was individually infected with virus or exposed to polybrene alone. Shake of cells from 2-4 similarly treated mixed glial cultures were then pooled to obtain approximately 5 million microglial cells in one 10-cm tissue culture plate that was used for RNA extraction and cRNA production.

Total RNA from primary microglial cultures was isolated 1 hour after plating using Trizol according to the manufacturer's instructions. For Affymetrix array analysis, approximately 5-10 million adherent microglia cells were used per isolation. Precipitated total RNA was stored as a pellet in 70% ethanol at -80C until it was solubilized for array and RT-PCR experiments. The integrity of RNA was evaluated with an RNA 6000 nano assay kit and Bioanalyzer 2100 (Agilent) to visualize and compare 18S and 28S rRNA bands prior to cDNA and cRNA probe synthesis.

The production of cRNA from total microglial RNA was performed by Genome Explorations using the MessageAmp aRNA Kit (Ambion) according to the manufacturer's instructions.

Mixed glia were grown in Advanced D-MEM (Gibco) supplemented with 5% fetal bovine serum (Hyclone), glutamine, and antibiotics (DMA5) until cells reached approximately 50% confluence (7-10 days in culture) at which time they were infected with the non-neurovirulent ecotropic murine leukemia virus Fr57E at a multiplicity of infection of ~1 infectious unit per cell in the presence of 8 µg/ml of polybrene. After 1 hour of virus/polybrene exposure, medium was replaced with fresh DMA5 containing 0.5 ng/ml GM-CSF. Cultures were grown until they were confluent and abundant phase bright cells appeared growing above the monolayer. Microglia were isolated by shake off, plated for 1 hour on Corning 10-cm tissue culture plates, washed 3 times with PBS, and then extracted with TRIzol for RNA isolation according to the manufacturer's instructions.

Mixed glia were grown in Advanced D-MEM (Gibco) supplemented with 5% fetal bovine serum (Hyclone), glutamine, and antibiotics (DMA5) until cells reached approximately 50% confluence (7-10 days in culture) at which time they were treated with 8 µg/ ml of polybrene in parallel with cells being infected with murine leukemia virus. After 1 hour of polybrene exposure medium was replaced with fresh DMA5 containing  0.5 ng/ml GM-CSF. Cultures were grown until they were confluent and abundant phase bright cells appeared growing above the monolayer. Microglia were isolated by shake off, plated for 1 hour on Corning 10-cm tissue culture plates, washed 3 times with PBS, and then extracted with TRIzol for RNA isolation according to the manufacturer's instructions.

The cRNA pellet was resuspended in 10 ul RNase-free H2O and 10.0 µg was fragmented by ion-mediated hydrolysis at 95C for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mM potassium acetate, 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16 hr at 45C to GeneChip MOE430A 2.0, which detects over 14,000 mouse annotated transcripts and ESTs (Affymetrix). Arrays were washed at 25C with 6 X SSPE (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA + 0.01% Tween 20) followed by a stringent wash at 50C with 100 mM MES, 0.1 M [Na+], 0.01% Tween 20.

The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes) and the fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix). The scanned images were analyzed using programs resident in GeneChip Operating System v1.2 (GCOS; Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to a constant target intensity of 250 for all arrays used. The expression data were analyzed as previously described (Lockhart et al, 1996, Nat. Biotechnol. 14:1675-1680). The signal intensity for each gene was calculated as the average intensity difference, represented by [(PM - MM)/(number of probe pairs)], where PM and MM denote perfect-match and mismatch probes.

Transcription profiling by array of mouse primary microglial cells infected with neurovirulent (FrCasE) and non-neurovirulent (Fr57E) virus

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