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Sample GSM283954 Query DataSets for GSM283954
Status Public on Apr 01, 2010
Title Donor 1 - CD34 - GCSF - mAdbID:81753
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 CD34 - GCSF Mobilized
Organism Homo sapiens
Characteristics Tissue: blood
Cell type: CD34
Treatment protocol Treatment type: compound
Agent: GCSF
Treatment dose: 10 ug / kg
Treatment time: 5 days
In-vivo treatment: Healthy people recieved GCSF for 5 days. 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: 3.25;; 4.3
Temperature: 32.19
Description mAdb experiment ID: 81753
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 2495 906 343 222 2598 828 357 169 0
184821_1 NULL 1 1 2 654 279 367 227 628 180 393 196 0
185589_1 -1.555680871 1 1 3 1073 397 351 226 2412 594 355 176 0
186357_1 NULL 1 1 4 4392 1364 352 207 3672 1120 335 162 0
187125_1 NULL 1 1 5 1187 454 359 205 1382 402 349 159 0
187893_1 NULL 1 1 6 485 217 369 208 553 185 335 156 -50
188661_1 NULL 1 1 7 798 337 358 198 1161 346 342 154 0
189429_1 NULL 1 1 8 449 226 333 206 500 172 337 155 -50
190197_1 NULL 1 1 9 467 217 329 218 1872 499 346 156 0
190965_1 2.507017612 1 1 10 3160 1163 354 212 818 272 343 150 0
273540_1 NULL 1 1 11 550 260 350 217 433 142 336 155 -50
274308_1 NULL 1 1 12 418 212 331 219 465 197 328 164 -50
275076_1 NULL 1 1 13 1549 672 338 233 1224 356 328 161 0
275844_1 NULL 1 1 14 4535 1472 326 216 4339 1204 324 144 0
276612_1 NULL 1 1 15 1760 539 319 189 522 228 339 145 0
277380_1 NULL 1 1 16 372 185 321 207 486 199 335 166 -50
278148_1 1.587536216 1 1 17 1671 698 319 209 734 282 335 149 0
278916_1 NULL 1 1 18 442 220 352 222 431 182 348 147 -50
279684_1 0.129953653 1 1 19 1654 719 354 222 1488 406 336 152 0
280452_1 0.629157364 1 1 20 907 430 354 211 536 226 335 150 0

Total number of rows: 17034

Table truncated, full table size 1000 Kbytes.




Supplementary file Size Download File type/resource
GSM283954.gpr.gz 1.8 Mb (ftp)(http) GPR
Processed data included within Sample table

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