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Sample GSM141398 Query DataSets for GSM141398
Status Public on Dec 19, 2006
Title PCA_25
Sample type RNA
 
Channel 1
Source name PCA_25
Organism Homo sapiens
Characteristics Prostate Carcinoma Sample 25
Extracted molecule total RNA
Extraction protocol Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using µCUT software MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Total RNA was isolated from captured cells with the RNAqueous Micro kit (Ambion) and treated with DNAse I according to the manufacturer's instructions. RNA quantification was perfomed using Ribogreen (Molecular Probes).
Label Cy5
Label protocol Total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 ?g per reaction. For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ?g), nuclease free water was added to 500 ?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ?l with nuclease free water. For labeling, 5 ?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ?l, 500 ?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ?l were used for analysis on a ND-1000 spectrophotometer.
 
Channel 2
Source name CPP
Organism Homo sapiens
Characteristics Clontech Prostate Pool
Extracted molecule total RNA
Extraction protocol commercially-obtained
Label Cy3
Label protocol Total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 ?g per reaction. For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ?g), nuclease free water was added to 500 ?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ?l with nuclease free water. For labeling, 5 ?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ?l, 500 ?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ?l were used for analysis on a ND-1000 spectrophotometer.
 
 
Hybridization protocol For hybridization, 40 ug of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 120 ?l of labeled cDNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 13,000 rcf for 6 min at RT. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18.6 ?l with nuclease free water and the following were added: 4 ?l of yeast tRNA (10 ?g/?l, Invitrogen, Carlsbad, CA), 4.9 ?l of 20X SSC and 0.84 ?l of 10% SDS. The probe was denatured at 100 Co for 3 min and centrifuged at 13,000 rcf for 45 sec. The probe was added directly to the microarray and a cover slip was added. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65 Co water bath. After hybridization, cover slips were removed by incubation in 2x SSC / 0.05% SDS. Slides were then washed in 2x SSC / 0.05% SDS for 5 min and 0.2x SSC / 0.05% SDS for five minutes. Slides were then washed in 0.2x SSC for 10 sec and centrifuged dry at 500 rpm for 5 min.
Scan protocol Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA).
Description See MIAME checklist, included in the Supplementary Methods, for additional information
Data processing Images were gridded and spots were quantified using GenePix Pro 4.0 software (Axon Instruments, Union City, CA). For all hybridizations, the log2 normalized Median of Ratios (as described below) was used.
 
Submission date Oct 20, 2006
Last update date Jul 22, 2008
Contact name Scott Tomlins
E-mail(s) tomlinss@med.umich.edu
Phone 734-615-1417
Organization name University of Michigan
Department Pathology
Lab Chinnaiyan Lab
Street address 1400 E. Medical Center Dr., 5410 CCGC
City Ann Arbor
State/province MI
ZIP/Postal code 48109
Country USA
 
Platform ID GPL2013
Series (1)
GSE6099 Integrative Molecular Concepts Modeling of Prostate Cancer Progression

Data table header descriptions
ID_REF
X the X-coordinate in um of the center of the feature indicator associated with the feature, where (0,0) is the top left of the image
Y the Y-coordinate in um of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image
Dia. the diameter in um of the feature indicator
F635.Median median feature pixel intensity at wavelength 1 (635 nm, Cy5)
F635.Mean mean feature pixel intensity at wavelength 1
F635.SD standard deviation of the feature pixel intensity at wavelength 1
B635.Median median feature background intensity at wavelength 1
B635.Mean mean feature background intensity at wavelength 1
B635.SD standard deviation of the feature background intenstiy at wavelength 1
X....B635.1SD percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 1
X....B635.2SD the percentage of fetaure pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 1
F635...Sat. the perentage of feature pixels at wavelength 1 that are saturated
F532.Median median feature pixel intensity at wavelength 2 (532 nm, Cy3)
F532.Mean mean featur pixel intensity at wavelength 2
F532.SD standard deviation of feature pixel intensity at wavelength 2
B532.Median medain feature backgroudn intensity at wavelength 2
B532.Mean mean feature background intensity at wavelength 2
B532.SD standard deviation of the feature background intensity at wavelength 2
X....B532.1SD the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 2
X....B532.2SD the percentage of feature pixels with intensities more than two standard deviations above the backroudnn pixel intensity, at wavelength 2
F532...Sat. the percentage of feature pixels at wavelength 2 that are saturated
Ratio.of.Medians..635.532. the ratio of the median intensities of each feature for each wavelength, with the median background subtracted
Ratio.of.Means..635.532. the ratio of the arithmetic mean intesnties of each feature for each wavelength, with the median background subtracted
Median.of.Ratios..635.532. the median of pixel by pixel ratios of pixel intensities, with the median backgroudn subtracted
Mean.of.Ratios..635.532. the arithemetic mean of the pixel by pixel ratios of pixel intensities, with the median background subtracted
Ratios.SD..635.532. the standard deviation of pixel intensity ratios
Rgn.Ratio..635.532. the regression ratio
Rgn.R. the coefficient of determination for the current regression value
F.Pixels the total number of feature pixels
B.Pixels the total number of background pixels
Sum.of.Medians the sum of hte median intensities for each wavelength, with the median background subtracted
Sum.of.Means the sum of the arithemetic mean intensities for each wavelength, with the median background subtracted
Log.Ratio..635.532. log (base 2) transform of the ratio of the medians
F635.Median...B635 the median feature pixel intenisty at wavelength 1 with the median background subtracted
F532.Median...B532 the median feature pixel intensity at wavelgnth 2 with the emdian background subtracted
F635.Mean...B635 the mean feature pixel intensity at wavelength 1 with the median background subtracted
F532.Mean...B532 the mean feature pixel inentisy at wavelength 2 with the median background subtracted
Flags 0 = unflagged; negative values indicate unalignend features flagged by GenePix or areas of obvious defects and were not used for data analysis
VALUE Normalized, log2-transformed Median of Ratios (635/532)

Data table
ID_REF X Y Dia. F635.Median F635.Mean F635.SD B635.Median B635.Mean B635.SD X....B635.1SD X....B635.2SD F635...Sat. F532.Median F532.Mean F532.SD B532.Median B532.Mean B532.SD X....B532.1SD X....B532.2SD F532...Sat. Ratio.of.Medians..635.532. Ratio.of.Means..635.532. Median.of.Ratios..635.532. Mean.of.Ratios..635.532. Ratios.SD..635.532. Rgn.Ratio..635.532. Rgn.R. F.Pixels B.Pixels Sum.of.Medians Sum.of.Means Log.Ratio..635.532. F635.Median...B635 F532.Median...B532 F635.Mean...B635 F532.Mean...B532 Flags VALUE
Hs6-1-1-1 2220 18720 110 2150 2078 770 358 372 148 97 95 0 1964 1794 642 281 283 50 98 96 0 1.065 1.137 1.197 1.18 1.584 1.164 0.9 80 556 3475 3233 0.091 1792 1683 1720 1513 0 0.3536
Hs6-1-2-1 2400 18720 110 1821 1874 529 346 368 152 100 100 0 1868 1853 526 279 282 52 100 100 0 0.928 0.971 0.963 0.975 1.364 0.991 0.848 80 532 3064 3102 -0.107 1475 1589 1528 1574 0 0.2099
Hs6-1-3-1 2590 18720 90 453 468 191 337 353 148 40 23 0 670 604 235 275 280 53 80 75 0 0.294 0.398 0.518 0.488 3.55 0.788 0.113 52 344 511 460 -1.768 116 395 131 329 -50 null
Hs6-1-4-1 2750 18720 120 1088 1205 576 336 356 146 89 81 0 1253 1237 630 279 285 52 95 94 0 0.772 0.907 0.952 0.944 1.802 0.913 0.821 120 581 1726 1827 -0.373 752 974 869 958 0 null
Hs6-1-5-1 2930 18710 110 906 895 331 352 363 140 86 76 0 1090 1038 325 281 283 48 98 96 0 0.685 0.717 0.714 0.654 1.981 0.799 0.576 80 463 1363 1300 -0.546 554 809 543 757 0 null
Hs6-1-6-1 3090 18710 100 2528 2429 790 372 388 147 100 100 0 4237 3821 1196 277 281 52 100 100 0 0.544 0.58 0.59 0.573 1.292 0.584 0.919 80 409 6116 5601 -0.877 2156 3960 2057 3544 0 -0.1697
Hs6-1-7-1 3270 18710 120 10195 10438 5335 349 366 143 100 99 0 11808 12408 7120 278 284 54 100 100 0 0.854 0.832 0.835 0.864 1.196 0.805 0.984 120 577 21376 22219 -0.228 9846 11530 10089 12130 0 0.1739
Hs6-1-8-1 3450 18700 110 1750 1853 715 353 373 152 100 100 0 1878 1804 764 282 293 77 100 100 0 0.875 0.986 1.031 1.036 1.429 0.999 0.883 80 528 2993 3022 -0.192 1397 1596 1500 1522 0 0.4119
Hs6-1-9-1 3630 18700 110 3911 3731 1875 372 396 162 100 100 0 6059 5715 2921 285 299 84 100 100 0 0.613 0.619 0.605 0.607 1.255 0.616 0.961 80 533 9313 8789 -0.706 3539 5774 3359 5430 0 -0.2386
Hs6-1-10-1 3800 18710 110 1682 1732 580 373 390 152 100 98 0 1652 1529 423 290 293 55 100 100 0 0.961 1.097 1.122 1.063 1.427 1.176 0.841 80 465 2671 2598 -0.057 1309 1362 1359 1239 0 0.2237
Hs6-1-11-1 3980 18700 120 1630 1608 721 357 382 147 97 90 0 2626 2505 1235 301 306 59 100 99 0 0.548 0.568 0.564 0.563 1.475 0.559 0.884 120 590 3598 3455 -0.869 1273 2325 1251 2204 0 -0.3148
Hs6-1-12-1 4160 18700 110 1818 1767 626 382 394 154 96 95 0 1732 1647 638 293 301 59 96 96 0 0.998 1.023 1.048 1.079 1.522 1.043 0.832 80 394 2875 2739 -0.003 1436 1439 1385 1354 0 0.3537
Hs6-1-13-1 4330 18690 190 4554 4402 1062 381 398 156 99 98 0 5145 4844 1130 302 312 70 98 98 0 0.862 0.885 0.886 0.896 1.259 0.891 0.959 256 1412 9016 8563 -0.215 4173 4843 4021 4542 0 -0.06547
Hs6-1-14-1 4500 18700 100 3698 3585 1258 379 395 162 100 100 0 4407 4202 1395 285 298 76 100 100 0 0.805 0.818 0.823 0.809 1.287 0.83 0.944 80 354 7441 7123 -0.313 3319 4122 3206 3917 0 0.001833
Hs6-1-15-1 4670 18690 110 2912 2877 1099 365 381 151 98 97 0 3104 2882 1131 285 291 52 98 98 0 0.904 0.967 0.96 1.006 1.583 0.979 0.928 80 492 5366 5109 -0.146 2547 2819 2512 2597 0 0.02619
Hs6-1-16-1 4850 18690 100 1486 1540 564 355 373 149 98 92 0 1436 1317 441 287 293 51 96 96 0 0.984 1.15 1.141 1.125 1.535 1.225 0.83 80 440 2280 2215 -0.023 1131 1149 1185 1030 0 null
Hs6-1-17-1 5020 18690 100 3910 3653 1255 366 376 144 100 100 0 4024 3654 1255 282 290 57 100 100 0 0.947 0.975 0.976 0.995 1.295 0.984 0.945 80 441 7286 6659 -0.078 3544 3742 3287 3372 0 0.1557
Hs6-1-18-1 5190 18690 90 1034 1082 347 365 381 146 96 94 0 1416 1295 371 288 295 61 98 98 0 0.593 0.712 0.762 0.71 1.844 0.759 0.701 52 342 1797 1724 -0.754 669 1128 717 1007 0 null
Hs6-1-19-1 5370 18680 120 2637 3355 2069 371 385 159 100 99 0 2342 3791 2527 296 310 90 100 100 0 1.108 0.854 0.841 0.875 1.361 0.836 0.954 120 575 4312 6479 0.147 2266 2046 2984 3495 0 0.03888
Hs6-1-20-1 5550 18680 120 2356 2556 1112 377 391 163 99 99 0 1928 2244 1170 301 314 93 99 99 0 1.216 1.121 1.162 1.197 1.369 1.076 0.927 120 588 3606 4122 0.283 1979 1627 2179 1943 0 0.2702

Total number of rows: 20000

Table truncated, full table size 3730 Kbytes.




Supplementary file Size Download File type/resource
GSM141398.gpr.gz 2.0 Mb (ftp)(http) GPR
Processed data included within Sample table

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