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Sample GSM32771 Query DataSets for GSM32771
Status Public on Nov 03, 2004
Title chip#12273516
Sample type RNA
 
Channel 1
Source name monkey C88015F pre-infected
Organism Macaca fascicularis
Extracted molecule total RNA
 
Channel 2
Source name monkey C88015F 2 wks post SHIV89.6P infection
Organism Macaca fascicularis
Extracted molecule total RNA
 
 
Description Experiment Design 1) Experiment Type normal vs diseased comparison, time course 2) Experimental Factor response to viral infection - SHIV89.6P 3) Number of hybridizations 60 4) Common Reference No 5) Quality Control Steps 4 technical replicate hybridizations per animal (3 replicate hybridizations for C89115M, C95067F) dual spotting of gene per array multiple spotting of select genes per array 2 biological replicates (2 different animals) per time point dye swaps 6) Experiment Description 4 cynomolgus monkeys (Macaca fascicularis) were infected with TCID50 of SHIV89.6P. Whole blood RNA was collected at 0, 1, 2, 4 and 5 weeks post-infection. Post-infection samples were competitively hybridized with pre-infected samples for each individual animal. Unpublished Data 7) Qualifier, value, source 2.2. Samples: samples used, extract preparation and labeling 2.2.1. Biosource properties 1) Organism (NCBI taxonomy) Macaca fascicularis 2) Contact details for sample Dr Erling W Rud Animal Resources Division, Health Canada. Sir Frederick Banting Research Centre Building 22, Room C308, postal locator 2203E Tunneys' Pasture Ottawa,Ontario, Canada K1A 0L2 Tel:(613) 957-8049 FAX:(613) 941-6625 erling_rud@hc-sc.gc.ca 3) Cell Type not applicable 4) Sex C88015F - female C89115M - male C94071M - male C95067F - female 5) Age C88015F - 13 years C89115M - 12 years C94071M - 7 years C95067F - 6 years 6) Developmental Stage adult 7) Organism Part peripheral whole blood 8) Strain or line not applicable 9) Genetic variation not applicable 10) Individual number C88015F, C89115M, C94071M & C95067F 11) Individual genetic characteristics not applicable 12) Disease state normal, AIDS 13) Targeted cell type blood 14) Cell line not applicable 2.2.2. Biomaterial manipulation 1) Growth conditions not applicable 2) In vivo treatments description - 4 cynomolgus monkeys (Macaca fascicularis) were infected intravenously with TCID50 of SHIV89.6P. 10 ml of peripheral blood was drawn at -2, 0, 1, 2, 4 and 5 weeks post-infection. 3) In vitro treatment not applicable 4) Treatment type infection 5) Compound not applicable 6) Separation technique not applicable 2.2.3. Hybridization extract preparation 1) Extraction method PAXgene peripheral blood RNA preparation 10 ml of peripheral blood was drawn from each animal directly into 4 PAXgene Blood RNA Tubes (QIAGEN #762125) according to manufacturer's protocol (http://www.qiagen.com/literature/Handbooks/PDF/DNA_RNA_isolation_clinical/INT/PAXgene_Blood_RNA/PaxgeneBloodRNAtube.pdf) Total RNA was purified using PAXgene Blood RNA kit (QIAGEN # 762134) according to manufacturer's protocol http://www.qiagen.com/literature/Handbooks/PDF/DNA_RNA_isolation_clinical/INT/PAXgene_Blood_RNA/1017455PAXgeneBloodRNA_prot.pdf) 2) Nucleic acid type total RNA 3) Amplification Method Total RNA was amplified using the Ambion MessageAmp aRNA kit (#1750), an Eberwine based method, with slight modifications to the manufacturer's protocol (http://www.transnet.ca/Amplification%20Protocol.htm). 2.2.3. Sample labeling 1) Amount of nucleic acid labeled 6 micrograms of aRNA was labeled with Cy3/Cy5 for hybridization 2) Label used Cy5 & Cy3 3) Label incorporation method aRNA was labelled by reverse transcription using the standard protocol of the Ontario Cancer Institute with slight modifications (http://www.transnet.ca/Hybridization%20Protocol_2.htm) 2.2.5. Spiking controls 1) Spiking control feature OCI Human 19k3-part1 OCI Human 19k3-part2 2) Spike type and qualifier not applicable 3) Qualifier, value, source not applicable 2.3. Hybridization procedure and parameters 2) Hybridization Protocol Protocol - (http://www.transnet.ca/Hybridization%20Protocol_2.htm) Solution Hybridization buffer: DIG solution (Roche cat.#1603558) supplemented with 0.45 ug/ul yeast tRNA, 0.45 ug/ul salmon sperm DNA Blocking agent not applicable Slide blocking not applicable Probe blocking: 0.45 ug/ul yeast tRNA, 0.45 ug/ul salmon sperm DNA during hybridization Wash procedure 3x15 min with 1xSSC, 0.1% SDS at 50 degrees Celsius, 3 fast washes in 1xSSC at 50 degrees Celsius Quantity of labelled target used all material generated from 6 ug amplified RNA per sample Time 18 hr (overnight) hybridization Concentration 0.2 ug/ul of aRNA per hybridization Volume 60 ul Temperature 30 degrees Celsius Description of the hybridization chamber hybridizations were incubated in a microscope slide box stored within a tissue culture incubator Qualifier, value, source not applicable 2.4. Measurements 2.4.1. Raw data Scanning protocol Scanning hardware: Scanarray Express Microarray Scanner, Packard Biosciences Scanning software: Scanarray Express Microarray Analysis System v1.1 Build #9 Scan Parameters Laser power: see individual image files in section 2.4.1 Spatial resolution:10 microns PMT gain: 60% 2.4.2. Image analysis and quantitation Image analysis software: Quantarray v.3.0, Packard BioSciences Availability: No longer commercially available Quantitation Parameters: Array Pattern: Spot rows: 25 Spot columns: 24 Spot spacing (vertical): 170 microns Spot spacing (horizontal): 170 microns Interstitial spacing: Off Array rows: 8 Array columns: 4 Array spacing (vertical): 4500 microns Array spacing (horizontal): 4500 microns Auto-adjustable Grid: Grid elasticity: 50/100 Spot elasticity: 50/100 Quantitation: Method: Fixed Circle Crosstalk correction: No Normalized to brightest: No Background subtraction: No Gene database file: for OCI Human 19k3-part1- H19k3a.qa (attached) for OCI Human 19k3-part2- H19k3b.qa (attached) Fixed circle quantitation parameters: Spot diameter: 60 microns Inner background diameter: 180 microns Outer background diameter: 200 microns Signal low percentile: 45 Signal high percentile: 95 Background low percentile: 5 Background high percentile: 55 Quantification output: Mean Intensity Quality Criteria Confidence calculation: Minimum Tolerance and weight: None selected Replicates: Combine replicates: no 2.4.3. Normalized and summarized data Data processing protocol Background subtraction and normalization Background subtraction and normalization was performed using the Quantarray Normalization Excel macro on individual quantitation output files. Each channel was background subtracted using the equation: ch Bkg. Subtr. Intensity = ch intensity - ch Bkg. Normalization strategy: total array Normalization algorithm: log ratio median centering Median log ratio = median(log2(ch1 Bkg. Subtr. Intensity) - log2(ch2 Bkg. Subtr. Intensity)) Normalization factor = 2^ median log ratio Normalized ch2 intensity = ch2 intensity*Normalization factor Statistical Analysis The background subtracted normalized intensities were used for statistical analysis. All spot intensities for within-chip, repeat hybridization, fluor-flip hybridization and biological replicates for each time point post-infection were compiled and tested for significance testing using the software package ArrayStat v.1.0, Imaging Research. ArrayStat parameters: Model selection: Manual Model selected: Proportional model with offsets Outlier detection: yes Threshold detection: Manual Threshold detected: p<0.01 Minimum No. of replicates: 12 (for 1 & 5 wks PI), 11 (for 2 & 4 wks PI) Conditions: Dependent Random Error Estimation Method: Small Sample Test: t-test Normalization: By median Nominal alpha: 0.05 Multiple Test correction: False Discovery Rate

Keywords = SHIV89.6P
Keywords = HIV
Keywords = nonhuman primate
Keywords = cDNA microarray
Keywords = time course
Lot batch = lot #464
 
Submission date Oct 20, 2004
Last update date May 27, 2005
Contact name Steven Edward Bosinger
E-mail(s) sbosinge@uhnres.utoronto.ca
Phone 416-340-4800x6965
Fax 416-340-3619
URL http://www.transnet.ca
Organization name Toronto General Research Institute, University Health Network
Department Division of Experimental Therapeutics
Lab Laboratory of host responses
Street address 200 Elizabeth St.
City Toronto
State/province Ontario
ZIP/Postal code M5G 2C1
Country Canada
 
Platform ID GPL350
Series (1)
GSE1854 Gene Expression Profiling of Host Response in Models of Acute HIV infection

Data table header descriptions
ID_REF
VALUE Log10 ratio of CH2/CH1
NormCH1 CH1 intensity after median normalization and bkg subtraction
NormCH2 CH2 intensity after median normalization and bkg subtraction

Data table
ID_REF VALUE NormCH1 NormCH2
1 -0.182942328 185.383336 121.6545524
2 -0.014352096 182.508336 176.5755672
3 0.107191516 352.799988 451.5647972
4 0.182670594 290.908336 443.0234961
5 -0.013551079 49.391665 47.87431655
6 -0.065437452 43.691667 37.58037963
7 -0.302696406 65.941669 32.84456609
8 0.103168131 29.383332 37.26225839
9 -0.101459918 65.583331 51.91986476
10 0.576158297 20.375 76.78138009
11 0.179555835 154.125 233.0391696
12 0.103023505 162.741669 206.3109462
13 0.243685847 41.200001 72.20762712
14 0.38552882 22.558332 54.8069714
15 -0.131552608 31.008333 22.90476348
16 -0.509173025 73.841664 22.86274909
17 -0.14442525 49.016666 35.14944875
18 0.36914359 21.016665 49.17081335
19 -0.036240528 3234.424968 2975.476691
20 -0.075563284 3545.800049 2979.551921

Total number of rows: 19200

Table truncated, full table size 742 Kbytes.




Supplementary data files not provided

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