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Sample GSM32400 Query DataSets for GSM32400
Status Public on Nov 03, 2004
Title chip#12243916
Sample type RNA
 
Channel 1
Source name monkey C88015F pre-infected
Organism Macaca fascicularis
Extracted molecule total RNA
 
Channel 2
Source name monkey C88015F 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Unpublished Data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
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 = Array lot 399
 
Submission date Oct 15, 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 -0.205039733 129.066666 80.49601097
2 -0.575856731 119.73333 31.79496348
3 -0.244007588 43.066666 24.55464524
4 -0.222075127 35.799999 21.46880576
5 0.625542154 14 59.11125629
6 0.789948272 6.933333 42.7454931
7 -0.657771628 92.933334 20.43618797
8 -0.003745306 29.200001 28.94926626
9 -0.059465315 30.333334 26.45177576
10 0.57137114 24.6 91.68667997
11 -0.449267963 35 12.43941858
12 -0.310030776 48.666668 23.83421413
13 -0.154997523 30.333334 21.22866206
14 -0.323085693 47.599998 22.6214918
15 -0.155270639 593.400024 415.0275436
16 -0.280598804 604.133362 316.6168442
17 -0.268374555 141.066666 76.0413551
18 -0.427010603 172.066666 64.37039004
19 -0.106591088 3467.733398 2713.030104
20 -0.059563739 4210.733398 3671.081325

Total number of rows: 19200

Table truncated, full table size 738 Kbytes.




Supplementary data files not provided

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