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Sample GSM141380 Query DataSets for GSM141380
Status Public on Dec 19, 2006
Title PCA_8
Sample type RNA
 
Channel 1
Source name PCA_8
Organism Homo sapiens
Characteristics Prostate Carcinoma Sample 8
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 1390 18500 110 3011 2903 1046 410 431 178 100 98 0 2984 2768 946 322 332 71 100 100 0 0.977 1.019 1.02 1.012 1.323 1.04 0.923 80 561 5263 4939 -0.033 2601 2662 2493 2446 0 0.585
Hs6-1-2-1 1570 18500 100 1000 1104 422 425 443 192 87 67 0 1845 1731 648 332 342 99 100 100 0 0.38 0.485 0.48 0.441 2.055 0.51 0.602 80 470 2088 2078 -1.396 575 1513 679 1399 0 null
Hs6-1-3-1 1760 18500 90 719 775 322 385 411 171 73 50 0 1148 1092 291 330 342 82 98 98 0 0.408 0.512 0.449 0.407 2.786 0.614 0.329 52 348 1152 1152 -1.292 334 818 390 762 0 null
Hs6-1-4-1 1910 18500 100 1164 1210 375 385 417 188 98 90 0 2250 2072 713 327 334 87 98 98 0 0.405 0.473 0.464 0.47 1.866 0.447 0.682 80 463 2702 2570 -1.304 779 1923 825 1745 0 0.03611
Hs6-1-5-1 2090 18500 100 774 814 330 389 411 170 81 58 0 1493 1309 508 325 328 75 97 91 0 0.33 0.432 0.392 0.398 2.264 0.454 0.463 80 440 1553 1409 -1.601 385 1168 425 984 0 null
Hs6-1-6-1 2260 18500 100 2266 2217 926 394 421 172 98 97 0 4074 3594 1574 336 344 74 100 100 0 0.501 0.56 0.589 0.568 1.417 0.55 0.89 80 435 5610 5081 -0.998 1872 3738 1823 3258 0 0.2802
Hs6-1-7-1 2440 18510 120 11653 11751 7182 395 423 181 99 98 0 14730 14677 8910 328 342 78 100 100 0 0.782 0.791 0.796 0.786 1.2 0.796 0.987 120 582 25660 25705 -0.355 11258 14402 11356 14349 0 0.0352
Hs6-1-8-1 2620 18500 100 1145 1185 539 405 417 168 91 75 0 1948 1828 849 327 333 73 100 100 0 0.457 0.52 0.524 0.533 1.804 0.53 0.708 80 435 2361 2281 -1.131 740 1621 780 1501 0 0.08698
Hs6-1-9-1 2790 18500 110 3265 3594 2356 407 432 192 97 96 0 5318 5606 3851 331 351 122 100 100 0 0.573 0.604 0.574 0.607 1.463 0.595 0.952 80 534 7845 8462 -0.803 2858 4987 3187 5275 0 -0.001444
Hs6-1-10-1 2970 18490 90 1299 1299 499 421 461 306 88 67 0 1826 1748 604 334 437 1267 61 0 0 0.588 0.621 0.61 0.53 2.369 0.25 0.529 52 363 2370 2292 -0.765 878 1492 878 1414 0 -0.02964
Hs6-1-11-1 3140 18500 100 1807 1834 756 432 463 281 97 83 0 3101 2787 1307 338 417 1088 77 53 0 0.498 0.572 0.572 0.579 1.683 0.378 0.682 80 494 4138 3851 -1.007 1375 2763 1402 2449 0 0.2353
Hs6-1-12-1 3330 18500 60 1016 1069 343 477 497 218 81 65 0 1954 1725 694 391 439 172 93 87 0 0.345 0.444 0.456 0.438 2.569 0.466 0.373 32 152 2102 1926 -1.536 539 1563 592 1334 0 null
Hs6-1-13-1 3500 18500 190 5604 5303 1597 406 428 178 100 100 0 7447 6826 1982 329 344 103 100 100 0 0.73 0.754 0.752 0.751 1.216 0.755 0.955 256 1392 12316 11394 -0.454 5198 7118 4897 6497 0 -0.0747
Hs6-1-14-1 3670 18500 90 581 653 338 371 410 173 59 32 0 434 558 389 327 336 95 53 28 0 1.963 1.221 1.839 1.515 4.941 1.184 0.3 52 298 317 513 0.973 210 107 282 231 -50 null
Hs6-1-15-1 3830 18500 100 3184 3130 1143 393 413 171 100 98 0 4144 4210 1522 318 324 84 100 100 0 0.729 0.703 0.703 0.696 1.299 0.703 0.926 80 440 6617 6629 -0.455 2791 3826 2737 3892 0 -0.004626
Hs6-1-16-1 4010 18500 100 1192 1211 447 396 418 183 93 81 0 1589 1456 609 324 327 71 97 95 0 0.629 0.72 0.735 0.753 2.039 0.729 0.69 80 452 2061 1947 -0.668 796 1265 815 1132 0 null
Hs6-1-17-1 4180 18500 100 4297 4373 2044 410 427 173 100 100 0 4490 4315 1978 327 336 94 100 100 0 0.934 0.994 1.02 0.999 1.328 1.01 0.95 80 466 8050 7951 -0.099 3887 4163 3963 3988 0 0.5639
Hs6-1-18-1 4340 18500 50 1234 1226 258 493 635 423 83 33 0 2018 2048 251 427 672 569 100 100 0 0.466 0.452 0.457 0.431 1.364 0.498 0.596 12 104 2332 2354 -1.102 741 1591 733 1621 0 -0.304
Hs6-1-19-1 4540 18500 120 1372 2067 1573 415 441 233 90 76 0 1520 3029 2730 324 385 267 90 82 0 0.8 0.611 0.613 0.647 2.088 0.591 0.939 120 691 2153 4357 -0.322 957 1196 1652 2705 0 null
Hs6-1-20-1 4720 18510 110 1237 1332 630 401 411 168 90 81 0 1749 2010 1102 314 325 83 100 97 0 0.583 0.549 0.533 0.515 2.191 0.53 0.74 80 466 2271 2627 -0.779 836 1435 931 1696 0 null

Total number of rows: 20000

Table truncated, full table size 3746 Kbytes.




Supplementary file Size Download File type/resource
GSM141380.gpr.gz 2.0 Mb (ftp)(http) GPR
Processed data included within Sample table

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