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Sample GSM283958 Query DataSets for GSM283958
Status Public on Apr 01, 2010
Title Donor 4 - CD133 - AMD3100 - mAdbID:81757
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 - AMD3100 Mobilized
Organism Homo sapiens
Characteristics Tissue: blood
Cell type: CD133
Treatment protocol Treatment type: compound
Agent: AMD3100
Treatment dose: 240ug / kg (AMD3100)
Treatment time: 12 hours
In-vivo treatment: Healthy 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: 3.23;; 3.97
Temperature: 30.11
Description mAdb experiment ID: 81757
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 3095 961 349 208 3034 882 544 278 0
184821_1 NULL 1 1 2 1021 416 354 207 1042 411 535 265 0
185589_1 -3.765141249 1 1 3 616 290 336 192 3376 811 535 291 0
186357_1 NULL 1 1 4 3451 1171 337 217 4000 1319 549 269 0
187125_1 NULL 1 1 5 1648 542 344 209 1765 669 532 248 0
187893_1 NULL 1 1 6 532 284 365 205 665 306 525 258 -50
188661_1 NULL 1 1 7 1078 391 342 202 1678 496 540 265 0
189429_1 NULL 1 1 8 559 268 334 212 649 301 541 290 -50
190197_1 NULL 1 1 9 687 332 350 218 5137 975 553 285 0
190965_1 2.999209404 1 1 10 6446 1645 345 219 1124 430 521 280 0
273540_1 -0.183047175 1 1 11 764 345 373 210 861 331 533 261 0
274308_1 NULL 1 1 12 607 275 347 222 711 338 535 265 -50
275076_1 NULL 1 1 13 2139 566 343 212 1517 468 538 266 0
275844_1 NULL 1 1 14 11535 8318 376 214 3494 2328 554 282 0
276612_1 NULL 1 1 15 2068 1361 373 206 751 350 517 272 0
277380_1 NULL 1 1 16 526 293 359 237 700 294 532 273 -50
278148_1 1.024603844 1 1 17 2048 822 426 393 1140 552 579 283 0
278916_1 NULL 1 1 18 551 240 395 291 671 303 533 292 -50
279684_1 0.685602427 1 1 19 2868 907 366 217 1760 561 546 292 0
280452_1 0.397877276 1 1 20 922 467 398 263 763 357 574 321 0

Total number of rows: 17034

Table truncated, full table size 1011 Kbytes.




Supplementary file Size Download File type/resource
GSM283958.gpr.gz 1.8 Mb (ftp)(http) GPR
Processed data included within Sample table

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