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Sample GSM225983 Query DataSets for GSM225983
Status Public on Aug 01, 2008
Title C40 vs. pLRT - repeat 4 - mAdbID:51262
Sample type RNA
 
Channel 1
Source name pLRT - Cy5
Organism Homo sapiens
Characteristics Cell line: HME16C human mammary epithelial cells
Cell type: hTERT immortalized human mammary epithelial cells
Other: These cells were infected with the control pLRT retroviral expression vector that does not express a gene product.
Treatment protocol Treatment type: compound
Agent: Doxycycline
Treatment dose: 250 ng/ml
Treatment time: 72 hours
Extracted molecule total RNA
Extraction protocol Trizol Extraction
Extraction method: guanidium HCL/phenol-chloroform
Other: Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA).
Label cy5
Label protocol Cy5 Labeling Protocol
Other: cDNA was labeled with Cy5 monofunctional reactive dye to amino allyl-modified dUTP incorporated into cDNA using the FairPlay Microarray labeling kit (Stratagene, La Jolla, CA).
 
Channel 2
Source name C40 - Cy3
Organism Homo sapiens
Characteristics Cell line: HME16C human mammary epithelial cells
Cell type: hTERT immortalized human mammary epithelial cells
Other: These cells were infected with the pLRT-H-RasV12C40 retroviral expression vector that directs the doxycycline-inducible expression of activated RasV12C40.
genotype/variation: RasV12C40
Treatment protocol Treatment type: compound
Agent: Doxycycline
Treatment dose: 250 ng/ml
Treatment time: 72 hours
Extracted molecule total RNA
Extraction protocol Trizol Extraction
Extraction method: guanidium HCL/phenol-chloroform
Other: Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA).
Label cy3
Label protocol Cy3 Labeling Protocol
Other: cDNA was labeled with Cy3 monofunctional reactive dye to amino allyl-modified dUTP incorporated into cDNA using the FairPlay Microarray labeling kit (Stratagene, La Jolla, CA).
 
 
Hybridization protocol NCI cDNA Microarray Hybridization
Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Add 1 µl COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 20 ul of 2X F-hyb buffer to samples. Load 40 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry. (Refer to "NCI Microarray Manual")
Scan protocol Creator: GenePix Pro 4.0.0.54
Scanner: GenePix 4000A [47890]
ScanPower: 100;; 100;; n/a;; n/a
LaserPower: 1.25;; 1.91;; n/a;; n/a
Temperature: 36.56
Description mAdb experiment ID: 51262
Data processing Data Processing Protocol
Calculation Method: Signal intensities of microarray features were calculated by subtracting the median local background from the median signal intensity. Features were considered for analysis if in the range 50-300ìm with signal intensity greater than 100 in both channels that were greater than one standard deviation above background with at least a 2:1 signal to background ratio. Signal intensities for an entire microarray were normalized to the 50% percentile median value, to correct for superior cy3 or cy5 labeling of one sample versus the other, or to superior cDNA synthesis of one sample versus the other. After filtering and normalization, the cy3 and cy5 values for the pLRT control cDNA and the experimental (V12 or effector domain) cDNA were expressed as a ratio to indicate the fold up- or down-regulation relative to the pLRT control cDNA.
 
Submission date Aug 30, 2007
Last update date Aug 14, 2011
Contact name Kathleen Kelly
E-mail(s) kk62j@nih.gov
Phone 3014354652
Fax 3014354655
Organization name National Institutes of Health
Department Cancer and Cell Biology Branch
Lab Kelly
Street address 37 Convent Drive
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL1528
Series (1)
GSE8916 Gene expression changes with inducible expression of activated ras and ras effector domain mutants

Data table header descriptions
ID_REF NCI mAdb well id plus replicate number
VALUE same as UNF_VALUE but with flagged values removed, experimental/control
PRE_VALUE 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
UNF_VALUE log ratio (log2 of PRE_VALUE)

Data table
ID_REF VALUE PRE_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 UNF_VALUE
1173588_1 0.913 1.883 1 1 1 937 465 273 272 748 305 270 112 0 0.913
1174364_1 -0.502 0.706 1 1 2 2535 1077 305 216 4544 1046 263 95 0 -0.502
1175152_1 0.991 1.988 1 1 3 1097 573 280 421 820 293 263 117 0 0.991
1175940_1 0.265 1.202 1 1 4 1175 417 265 358 1290 327 264 99 0 0.265
1176716_1 -0.265 0.832 1 1 5 2097 818 287 295 3214 843 267 119 0 -0.265
1177504_1 -0.072 0.952 1 1 6 2222 796 301 270 2997 804 261 154 0 -0.072
1178580_1 -0.029 0.980 1 1 7 2757 1070 297 307 3664 1124 263 133 0 -0.029
1179356_1 1.208 2.311 1 1 8 596 383 284 247 459 132 276 166 0 1.208
1180144_1 -0.288 0.819 1 1 9 835 659 285 291 1175 383 265 252 0 -0.288
1180932_1 0.816 1.760 1 1 10 704 295 265 276 592 190 254 194 0 0.816
1181708_1 0.145 1.105 1 1 11 645 322 260 245 726 246 254 147 0 0.145
1182496_1 0.746 1.678 1 1 12 583 275 276 211 509 146 261 113 0 0.746
1183572_1 0.115 1.083 1 1 13 712 369 290 204 781 248 253 128 0 0.115
1184348_1 0.275 1.210 1 1 14 662 335 304 437 652 223 251 186 0 0.275
1185136_1 -0.677 0.626 1 1 15 1217 606 287 436 2275 628 260 209 0 -0.677
1185924_1 1.066 2.094 1 1 16 644 275 284 295 491 181 258 104 0 1.066
1186700_1 0.117 1.085 1 1 17 738 439 265 199 851 345 260 145 0 0.117
1187488_1 0.394 1.314 1 1 18 499 271 275 346 491 133 260 251 0 0.394
1188564_1 -0.442 0.736 1 1 19 800 447 284 309 1210 356 260 204 0 -0.442
1189340_1 -0.397 0.759 1 1 20 2480 940 274 260 4202 1397 264 147 0 -0.397

Total number of rows: 21794

Table truncated, full table size 1517 Kbytes.




Supplementary file Size Download File type/resource
GSM225983.gpr.gz 2.1 Mb (ftp)(http) GPR
Processed data included within Sample table

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