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Sample GSM141367 Query DataSets for GSM141367
Status Public on Dec 19, 2006
Title MET_HR_11
Sample type RNA
 
Channel 1
Source name MET_HR_11
Organism Homo sapiens
Characteristics Metastatic Prostate Carcinoma - Hormone Refractory Sample 11
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 1210 18680 100 894 924 481 136 140 45 96 93 0 886 749 362 94 97 23 100 98 0 0.957 1.203 1.188 1.204 1.609 1.262 0.883 80 471 1550 1443 -0.063 758 792 788 655 0 0.644
Hs6-1-2-1 1390 18670 100 656 642 275 134 141 43 97 93 0 811 734 306 95 99 25 100 97 0 0.729 0.795 0.76 0.772 1.58 0.812 0.862 80 447 1238 1147 -0.456 522 716 508 639 0 0.2307
Hs6-1-3-1 1560 18670 80 144 139 46 137 143 42 13 5 0 191 181 94 94 96 22 69 63 0 0.072 0.023 0.354 0.331 3.69 0.159 0.011 52 298 104 89 -3.793 7 97 2 87 -50 null
Hs6-1-4-1 1720 18670 100 1602 1517 700 135 141 49 98 97 0 1213 1147 527 91 96 27 97 97 0 1.307 1.309 1.296 1.328 1.387 1.314 0.931 80 485 2589 2438 0.387 1467 1122 1382 1056 0 1.039
Hs6-1-5-1 1900 18680 70 257 255 91 139 148 55 71 53 0 494 425 164 102 121 69 90 84 0 0.301 0.359 0.353 0.385 1.837 0.378 0.526 32 231 510 439 -1.732 118 392 116 323 0 null
Hs6-1-6-1 2070 18670 110 1079 1016 381 136 142 48 98 97 0 1926 1714 630 96 105 48 100 100 0 0.515 0.544 0.54 0.534 1.381 0.54 0.923 80 517 2773 2498 -0.957 943 1830 880 1618 0 -0.1474
Hs6-1-7-1 2260 18680 120 3113 3702 2222 129 133 38 100 100 0 4297 4714 2486 97 99 24 100 100 0 0.71 0.774 0.763 0.751 1.314 0.805 0.941 120 543 7184 8190 -0.493 2984 4200 3573 4617 0 -0.2743
Hs6-1-8-1 2430 18670 110 674 627 292 132 136 40 98 91 0 991 853 411 94 97 23 100 100 0 0.604 0.652 0.662 0.629 1.476 0.67 0.894 80 462 1439 1254 -0.727 542 897 495 759 0 0.07276
Hs6-1-9-1 2590 18670 110 1952 1894 905 135 141 46 100 100 0 3144 2712 1279 96 101 42 100 100 0 0.596 0.672 0.702 0.661 1.331 0.669 0.96 80 496 4865 4375 -0.746 1817 3048 1759 2616 0 -0.07681
Hs6-1-10-1 2770 18680 110 550 500 231 134 141 50 86 80 0 741 614 322 94 104 51 82 78 0 0.643 0.704 0.718 0.806 2.236 0.708 0.886 80 485 1063 886 -0.637 416 647 366 520 0 -0.02608
Hs6-1-11-1 2960 18680 120 906 1073 720 133 140 48 97 95 0 1026 1311 878 94 102 46 98 95 0 0.829 0.772 0.766 0.77 1.428 0.789 0.949 120 566 1705 2157 -0.270 773 932 940 1217 0 0.4042
Hs6-1-12-1 3120 18680 100 379 374 157 130 131 39 88 77 0 740 655 309 91 96 34 95 93 0 0.384 0.433 0.458 0.379 2.371 0.432 0.782 80 313 898 808 -1.382 249 649 244 564 0 -0.3619
Hs6-1-13-1 3310 18680 210 3325 3502 1403 129 136 108 100 100 0 3173 3131 983 90 98 112 100 100 0 1.037 1.109 1.067 1.084 1.26 1.163 0.945 316 1473 6279 6414 0.052 3196 3083 3373 3041 0 0.09048
Hs6-1-14-1 3490 18680 80 153 210 146 130 157 270 11 0 0 136 280 279 90 119 291 23 15 0 0.5 0.421 0.639 0.83 3.327 0.843 0.858 52 202 69 270 -1.000 23 46 80 190 -50 null
Hs6-1-15-1 3640 18680 120 657 692 327 129 143 173 85 66 0 1071 1102 589 89 103 184 93 80 0 0.538 0.556 0.566 0.583 1.623 0.546 0.918 120 510 1510 1576 -0.895 528 982 563 1013 0 -0.4118
Hs6-1-16-1 3810 18670 110 380 372 135 124 128 38 92 88 0 640 556 246 91 93 20 95 93 0 0.466 0.533 0.522 0.552 1.801 0.505 0.774 80 481 805 713 -1.101 256 549 248 465 0 null
Hs6-1-17-1 4000 18670 110 941 885 444 123 126 38 93 90 0 2069 1713 882 91 93 22 100 98 0 0.414 0.47 0.471 0.47 1.641 0.469 0.927 80 509 2796 2384 -1.274 818 1978 762 1622 0 -0.7661
Hs6-1-18-1 4170 18670 110 435 418 171 125 129 38 88 87 0 584 556 279 90 91 21 91 88 0 0.628 0.629 0.633 0.701 2.177 0.605 0.814 80 486 804 759 -0.672 310 494 293 466 0 0.04366
Hs6-1-19-1 4350 18680 120 1647 2460 2357 129 187 522 90 67 0 1040 1692 1329 89 178 859 55 37 0 1.596 1.454 1.394 1.502 1.395 1.669 0.875 120 547 2469 3934 0.675 1518 951 2331 1603 0 0.9597
Hs6-1-20-1 4530 18680 120 807 976 601 126 184 530 59 33 0 900 1012 618 90 183 872 46 14 0 0.841 0.922 0.9 0.927 1.528 0.896 0.927 120 531 1491 1772 -0.250 681 810 850 922 0 0.22

Total number of rows: 20000

Table truncated, full table size 3566 Kbytes.




Supplementary file Size Download File type/resource
GSM141367.gpr.gz 1.9 Mb (ftp)(http) GPR
Processed data included within Sample table

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