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Sample GSM283955 Query DataSets for GSM283955
Status Public on Apr 01, 2010
Title Donor 1 - CD133 - GCSF plus AMD3100 - mAdbID:81754
Sample type RNA
 
Channel 1
Source name Pooled Normal Donor PBMC
Organism Homo sapiens
Characteristics Tissue: blood
Cell type: PBMC
Extracted molecule total RNA
Extraction protocol RNA extraction with TRIZOL
Extraction method: TRIZOL
Other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturers instructions. RNA integrity was assessed by the presence of 28S and 18S bands using Agilent bioanalyzer and quantified using nanodrop with A260/A280 ratios > 2.0 after dilution in DEPC water.
Label cy3
Label protocol Cy3 Sample Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy3) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
 
Channel 2
Source name CD133 - GCSF plus AMD3100 Mobilized
Organism Homo sapiens
Characteristics Tissue: blood
Cell type: CD133
Treatment protocol Treatment type: compound
Agent: GCSF plus AMD3100
Treatment dose: 10 ug / kg (GCSF); 240ug / kg (AMD3100)
Treatment time: 5 days (GCSF); 12 hours (AMD3100)
In-vivo treatment: Healthy donors recieved GCSF for 5 days. On the 5th day donors recieved one dose of AMD3100. Then the PBSC were collected by aphoresis.
Extracted molecule total RNA
Extraction protocol RNA extraction with TRIZOL
Extraction method: TRIZOL
Other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturers instructions. RNA integrity was assessed by the presence of 28S and 18S bands using Agilent bioanalyzer and quantified using nanodrop with A260/A280 ratios > 2.0 after dilution in DEPC water.
Label cy5
Label protocol Cy5 Sample Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy5) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
 
 
Hybridization protocol Sample Hybrization Protocol
Wash procedure: Washing: 1. Wash with 2x SSC + 0.1% SDS to remove coverslip. 2. Wash with 1x SSC for 1 min. 3. Wash with 0.2x SSC for 1 min. 4. Wash with 0.05x SSC for 10-20 seconds. 5. Centrifuge slide at 80-100 g for 3 min.
Other: Hybridization: Combine Cy3 and Cy5 labeled target (adjust the color to purple) and complete dry the sample by speed vacuum . Add 37ul of the following mixture to the dried target (for 20x40mm slide): 1ul 50x Denhardt’s blocking solution (Sigma Cat# 2532) 1ul poly dA (8mg/ml Pharmacia Cat# 27-7988-01) 1ul yeast tRNA (4mg/ml Sigma Cat# R8759) 10ul Human Cot I DNA (1mg/ml Gibco BRL Cat# 15279-011) 3ul 20X SSC 1ul 10% SDS 20ul of DEPC H2O Heat for 2 min at 99oC and Cool to RT. Apply target mixture to array slide, add coverslip, place in humidified hyb chamber, and hybridize at 65oC over night.
Scan protocol Creator: GenePix Pro 4.0.1.17
Scanner: GenePix 4000B [84945]
ScanPower: 10;; 10
LaserPower: 3.27;; 4.68
Temperature: 32.95
Description mAdb experiment ID: 81754
Data processing BRBArrayTools
Calculation Method: Fluorescence intensities generated by Cy5 and Cy3 probes hybridized onto the microarray slides were scanned were scanned on a GenePix 4000 (Axon Instruments, Union City, CA) at variable PMT voltage to obtain maximal signal intensities with < 1% probe saturation. Data were uploaded to National Institute of Health, NCI/CCR µArray database (http://nciarray.nci.nih.gov). Raw data were retrieved in BRB-ArrayTools(http://linus.nci.nih.gov/BRBArrayTools.html) format for analysis and normalization. Following filter criteria was applied to the data set: 1. spots were excluded if intensity < 300, and spot size less than 25um 2. genes were filtered out if >80% of experiment with missing data values. 3. log2 transformed data were normalized using lowess smoother, and intensity ratios were truncated if greater than 64. Transcriptional data were process by averaging results in replicate experiments for the same genes. Hierchical clustering analysis was performed on BRBArray tools using centered correlation as similarity metric and centroid linkage method: Cluster 3:0. Heat maps were generated using Eisen’s TreView Software.
 
Submission date Apr 23, 2008
Last update date Mar 23, 2009
Contact name Francesco Maria Marincola
E-mail(s) fmarincola@sidra.org
Phone 301-793-8210
Organization name Sidra Medical and Research Center
Street address Al Nasr Tower, AL Corniche Street, PO Box 26999
City Doha
ZIP/Postal code PO Box 26999
Country Qatar
 
Platform ID GPL5959
Series (2)
GSE11247 Peripheral blood stem cell gene profiling
GSE11255 Peripheral blood stem cell gene and microRNA profiling

Data table header descriptions
ID_REF NCI mAdb well id plus replicate number
VALUE log2 of the Calibrated Ratio (Cy5 channel/Cy3 channel)
Slide_block Array block location
Slide_column Array column location
Slide_row Array row location
CY5_mean Red Channel Sample mean Signal (Background Subtracted)
CY5_SD Red Channel Sample Standard Deviation
CY5_BKD_median Red Channel Sample median Background Level
CY5_BKD_SD Red Channel Sample Background Standard Deviation
CY3_mean Green Channel Sample mean Signal (Background Subtracted)
CY3_SD Green Channel Sample Standard Deviation
CY3_BKD_median Green Channel Sample median Background Level
CY3_BKD_SD Green Channel Sample Background Standard Deviation
Flag Quality flag 0->good, -50->Not found, -100->Bad

Data table
ID_REF VALUE Slide_block Slide_column Slide_row CY5_mean CY5_SD CY5_BKD_median CY5_BKD_SD CY3_mean CY3_SD CY3_BKD_median CY3_BKD_SD Flag
184053_1 NULL 1 1 1 2501 664 197 117 2481 597 590 220 0
184821_1 NULL 1 1 2 331 176 196 113 664 230 595 213 -50
185589_1 -3.101853848 1 1 3 235 119 192 114 2379 546 559 206 0
186357_1 NULL 1 1 4 2178 671 179 110 3634 1067 532 211 0
187125_1 NULL 1 1 5 1012 346 166 107 1312 404 522 225 0
187893_1 NULL 1 1 6 208 111 173 103 583 212 536 216 -50
188661_1 NULL 1 1 7 509 196 185 112 1169 326 542 196 0
189429_1 NULL 1 1 8 221 122 185 112 620 216 554 202 -50
190197_1 NULL 1 1 9 276 156 182 106 1992 551 541 211 0
190965_1 2.455029726 1 1 10 3407 954 185 118 932 306 552 207 0
273540_1 NULL 1 1 11 339 170 183 130 620 247 552 211 -50
274308_1 NULL 1 1 12 279 137 174 122 611 185 563 210 -50
275076_1 NULL 1 1 13 1460 664 179 112 1131 343 565 228 0
275844_1 NULL 1 1 14 9204 3316 179 119 3404 1044 539 222 0
276612_1 NULL 1 1 15 2227 759 174 112 653 226 537 203 0
277380_1 NULL 1 1 16 285 159 165 112 604 223 535 200 -50
278148_1 0.948461652 1 1 17 1099 505 186 140 725 283 555 214 0
278916_1 NULL 1 1 18 467 187 218 186 659 207 543 216 0
279684_1 0.489510924 1 1 19 2352 590 205 207 1589 434 541 217 0
280452_1 NULL 1 1 20 615 205 205 185 643 204 539 214 0

Total number of rows: 17034

Table truncated, full table size 1002 Kbytes.




Supplementary file Size Download File type/resource
GSM283955.gpr.gz 1.8 Mb (ftp)(http) GPR
Processed data included within Sample table

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