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Sample GSM141358 Query DataSets for GSM141358
Status Public on Dec 19, 2006
Title MET_HR_3a
Sample type RNA
 
Channel 1
Source name MET_HR_3a
Organism Homo sapiens
Characteristics Metastatic Prostate Carcinoma - Hormone Refractory Sample 3a
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 1610 18700 100 959 1075 559 748 779 330 41 30 0 1111 1123 660 1065 1076 207 36 33 0 4.587 5.638 0.933 0.911 3.127 1.199 0.428 80 512 257 385 2.198 211 46 327 58 -50 null
Hs6-1-2-1 1800 18700 70 1916 1992 670 856 901 418 87 78 0 1891 1847 657 1056 1122 300 78 62 0 1.269 1.436 1.337 1.548 3.479 1.282 0.65 32 225 1895 1927 0.344 1060 835 1136 791 0 -0.07041
Hs6-1-3-1 1960 18700 100 1054 1108 523 814 867 328 41 20 0 1022 1088 444 1139 1148 217 25 13 0 -2.051 -5.765 1.355 1.205 4.699 1.755 0.302 80 470 123 243 Error 240 -117 294 -51 -50 null
Hs6-1-4-1 2130 18700 100 1605 1600 699 767 796 300 78 61 0 1586 1560 707 1110 1069 228 57 50 0 1.761 1.851 1.393 1.616 2.714 1.308 0.597 80 440 1314 1283 0.816 838 476 833 450 0 null
Hs6-1-5-1 2310 18700 100 876 933 446 824 857 342 22 12 0 1001 918 512 1147 1153 314 16 3 0 -0.356 -0.476 0.767 0.765 3.686 0.876 0.36 80 420 -94 -120 Error 52 -146 109 -229 -50 null
Hs6-1-6-1 2470 18710 80 2215 2309 1077 934 1007 421 80 71 0 2597 2787 1368 1278 1333 340 82 78 0 0.971 0.911 0.907 0.774 2.102 0.801 0.824 52 304 2600 2884 -0.042 1281 1319 1375 1509 0 0.2399
Hs6-1-7-1 2650 18700 120 6772 8217 5581 982 1054 424 92 90 0 7765 8898 6037 1306 1394 351 91 90 0 0.896 0.953 0.945 0.927 1.462 0.939 0.98 120 601 12249 14827 -0.158 5790 6459 7235 7592 0 0.3149
Hs6-1-8-1 2830 18700 110 2817 3028 1628 1096 1158 451 82 75 0 2901 3008 1600 1509 1496 403 76 65 0 1.236 1.289 1.138 1.154 1.698 1.101 0.848 80 530 3113 3431 0.306 1721 1392 1932 1499 0 0.2552
Hs6-1-9-1 3010 18700 110 4483 4541 2516 1106 1116 416 82 78 0 5160 5365 2952 1519 1446 357 85 78 0 0.927 0.893 0.877 0.818 1.692 0.865 0.936 80 510 7018 7281 -0.109 3377 3641 3435 3846 0 0.1131
Hs6-1-10-1 3180 18700 100 1686 1712 868 1045 1060 405 57 41 0 1534 1512 822 1425 1380 345 37 28 0 5.881 7.667 1.292 1.195 3.788 1.346 0.579 80 463 750 754 2.556 641 109 667 87 -50 null
Hs6-1-11-1 3360 18700 110 2803 3101 1706 1008 1036 373 85 77 0 2775 2950 1573 1430 1350 319 75 68 0 1.335 1.377 1.278 1.321 2.136 1.181 0.858 80 545 3140 3613 0.416 1795 1345 2093 1520 0 0.6804
Hs6-1-12-1 3530 18710 100 1807 2075 1161 1051 1084 362 67 52 0 1612 1917 1135 1420 1384 285 45 38 0 3.938 2.06 1.284 1.267 2.665 1.224 0.742 80 474 948 1521 1.977 756 192 1024 497 -50 null
Hs6-1-13-1 3710 18700 110 3898 3980 1462 1041 1057 335 97 95 0 3944 4142 1544 1407 1383 236 95 91 0 1.126 1.075 1.097 1.144 1.674 1.026 0.878 80 510 5394 5674 0.171 2857 2537 2939 2735 0 0.06961
Hs6-1-14-1 3880 18700 110 2656 2912 1562 908 943 319 83 78 0 2461 2727 1506 1301 1233 287 77 71 0 1.507 1.405 1.225 1.142 3.213 1.152 0.84 80 510 2908 3430 0.592 1748 1160 2004 1426 0 0.1217
Hs6-1-15-1 4060 18710 100 1337 1465 811 933 989 371 52 37 0 1524 1551 952 1263 1228 316 47 31 0 1.548 1.847 0.946 1.001 2.196 0.928 0.619 80 434 665 820 0.630 404 261 532 288 -50 null
Hs6-1-16-1 4230 18710 100 1418 1476 839 975 1018 383 53 30 0 1480 1446 800 1281 1262 265 43 33 0 2.226 3.036 1.103 1.067 2.956 1.283 0.576 80 481 642 666 1.155 443 199 501 165 -50 null
Hs6-1-17-1 4410 18710 110 2767 2968 1684 958 975 330 83 78 0 2682 2825 1519 1252 1236 175 80 76 0 1.265 1.278 1.185 1.22 2.07 1.2 0.877 80 486 3239 3583 0.339 1809 1430 2010 1573 0 0.04149
Hs6-1-18-1 4560 18710 80 1678 1653 545 866 913 324 76 57 0 1648 1591 616 1177 1160 223 65 51 0 1.724 1.901 1.414 1.274 3.77 1.2 0.562 52 302 1283 1201 0.786 812 471 787 414 0 null
Hs6-1-19-1 4750 18710 120 5114 5320 2974 959 1050 838 87 80 0 4919 5250 3007 1220 1304 380 87 86 0 1.123 1.082 1.042 1.053 1.545 1.097 0.874 120 604 7854 8391 0.168 4155 3699 4361 4030 0 0.2813
Hs6-1-20-1 4930 18710 110 5363 5577 3096 1060 1134 473 95 87 0 3883 3917 1927 1344 1393 432 85 80 0 1.695 1.756 1.668 1.812 1.675 1.672 0.934 80 562 6842 7090 0.761 4303 2539 4517 2573 0 0.726

Total number of rows: 20000

Table truncated, full table size 3748 Kbytes.




Supplementary file Size Download File type/resource
GSM141358.gpr.gz 2.1 Mb (ftp)(http) GPR
Processed data included within Sample table

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