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Sample GSM32447 Query DataSets for GSM32447
Status Public on Nov 03, 2004
Title chip#12243930
Sample type RNA
Channel 1
Source name monkey C95067F pre-infected
Organism Macaca fascicularis
Extracted molecule total RNA
Channel 2
Source name monkey C95067F 5 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 Unpublished Data 7) Qualifier, value, source 2.2.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 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 ( Total RNA was purified using PAXgene Blood RNA kit (QIAGEN # 762134) according to manufacturer's protocol 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 ( 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 ( 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 - ( 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- (attached) for OCI Human 19k3-part2- (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 = Array lot 403
Submission date Oct 16, 2004
Last update date May 27, 2005
Contact name Steven Edward Bosinger
Phone 416-340-4800x6965
Fax 416-340-3619
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
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
1 -0.037810399 61.866664 56.70825709
2 0.195344569 55.024997 86.27889944
3 0.111260754 34.041665 43.98165299
4 0.069041142 31.716668 37.18165336
5 -0.112015075 16.875 13.03853228
6 -0.916373298 30.875001 3.743119403
7 0.351384957 12.583333 28.26055243
8 0.106862193 27.266666 34.87339515
9 -0.466082146 62.583335 21.39816623
10 0.288301067 18.758332 36.43302948
11 -0.006227877 22.466667 22.14678918
12 0.417110086 11.866667 31.00550728
13 -0.366509005 97.199997 41.79816792
14 0.030645347 36.625 39.30275374
15 -0.056596916 418.666656 367.5119435
16 0.04377119 285.066681 315.295411
17 0.64850773 14.266666 63.50826139
18 0.15022651 22.733334 32.1284403
19 -0.067096443 3528.783447 3023.629439
20 -0.062368527 3197.600098 2769.845989

Total number of rows: 19200

Table truncated, full table size 737 Kbytes.

Supplementary data files not provided

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