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Sample GSM141384 Query DataSets for GSM141384
Status Public on Dec 19, 2006
Title PCA_11b
Sample type RNA
 
Channel 1
Source name PCA_11b
Organism Homo sapiens
Characteristics Prostate Carcinoma Sample 11b
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 1560 18560 100 2201 2213 485 196 202 57 100 100 0 1719 1687 323 272 278 60 100 100 0 1.386 1.425 1.434 1.423 1.197 1.429 0.927 80 503 3452 3432 0.471 2005 1447 2017 1415 0 0.3146
Hs6-1-2-1 1730 18570 100 1927 1844 431 194 199 55 100 100 0 2339 2237 461 269 275 61 100 100 0 0.837 0.838 0.834 0.832 1.218 0.843 0.943 80 488 3803 3618 -0.256 1733 2070 1650 1968 0 -0.3288
Hs6-1-3-1 1920 18570 60 259 264 75 209 215 60 43 15 0 585 575 108 273 285 70 93 93 0 0.16 0.182 0.217 0.158 4.156 0.139 0.046 32 170 362 357 -2.642 50 312 55 302 0 null
Hs6-1-4-1 2070 18570 110 1453 1403 284 187 191 54 100 100 0 1771 1746 372 270 276 64 100 100 0 0.843 0.824 0.818 0.829 1.219 0.811 0.931 80 538 2767 2692 -0.246 1266 1501 1216 1476 0 -0.06375
Hs6-1-5-1 2240 18580 110 606 615 148 188 191 61 100 100 0 937 908 244 259 267 72 98 98 0 0.617 0.658 0.649 0.684 1.592 0.632 0.802 80 514 1096 1076 -0.698 418 678 427 649 0 null
Hs6-1-6-1 2420 18580 110 2465 2317 449 189 197 66 100 100 0 3253 3074 574 264 273 68 100 100 0 0.761 0.757 0.752 0.757 1.158 0.763 0.964 80 494 5265 4938 -0.393 2276 2989 2128 2810 0 -0.1757
Hs6-1-7-1 2590 18580 120 17236 16754 6734 193 198 61 100 100 0 11583 10701 4066 271 279 63 100 100 0 1.507 1.588 1.6 1.572 1.239 1.607 0.983 120 527 28355 26991 0.591 17043 11312 16561 10430 0 0.2948
Hs6-1-8-1 2750 18580 110 2192 2152 490 183 189 55 100 100 0 2461 2359 584 256 264 67 100 100 0 0.911 0.936 0.924 0.95 1.169 0.916 0.959 80 459 4214 4072 -0.134 2009 2205 1969 2103 0 -0.09604
Hs6-1-9-1 2930 18580 120 4848 4248 1817 180 187 51 100 99 0 5380 4770 2121 262 271 71 100 99 0 0.912 0.902 0.895 0.919 1.237 0.888 0.972 120 573 9786 8576 -0.133 4668 5118 4068 4508 0 -0.1865
Hs6-1-10-1 3100 18580 110 2121 2064 481 176 182 50 100 100 0 1944 1872 385 261 273 97 100 100 0 1.156 1.172 1.178 1.169 1.195 1.172 0.945 80 473 3628 3499 0.209 1945 1683 1888 1611 0 -0.06146
Hs6-1-11-1 3280 18590 120 2948 2696 1099 172 178 50 100 100 0 3140 2879 1142 253 266 64 99 99 0 0.962 0.961 0.96 0.975 1.232 0.96 0.972 120 551 5663 5150 -0.057 2776 2887 2524 2626 0 0.08165
Hs6-1-12-1 3440 18590 110 1850 1797 422 174 178 48 100 100 0 1988 1908 435 249 259 64 100 100 0 0.964 0.978 1.004 0.979 1.182 0.973 0.953 80 441 3415 3282 -0.053 1676 1739 1623 1659 0 -0.04131
Hs6-1-13-1 3620 18590 150 4701 4456 1123 176 180 53 100 100 0 4501 4098 1043 266 274 73 100 100 0 1.068 1.117 1.117 1.121 1.15 1.11 0.973 156 810 8760 8112 0.096 4525 4235 4280 3832 0 -0.2215
Hs6-1-14-1 3790 18590 110 5116 4793 1421 178 182 56 100 100 0 4509 4178 1176 251 260 67 100 100 0 1.16 1.175 1.17 1.164 1.201 1.184 0.964 80 445 9196 8542 0.214 4938 4258 4615 3927 0 0.04067
Hs6-1-15-1 3960 18600 120 4812 4483 1862 183 185 51 100 100 0 3110 2846 1126 257 263 64 100 100 0 1.623 1.661 1.675 1.671 1.237 1.668 0.97 120 539 7482 6889 0.698 4629 2853 4300 2589 0 0.4236
Hs6-1-16-1 4130 18600 100 2297 2193 545 178 182 49 100 100 0 1827 1718 440 253 257 57 100 100 0 1.346 1.375 1.384 1.403 1.224 1.37 0.946 80 427 3693 3480 0.429 2119 1574 2015 1465 0 null
Hs6-1-17-1 4300 18600 110 5300 4976 1367 178 184 52 100 100 0 4534 4339 1258 266 285 92 100 100 0 1.2 1.178 1.201 1.185 1.219 1.147 0.919 80 505 9390 8871 0.263 5122 4268 4798 4073 0 -0.01413
Hs6-1-18-1 4470 18610 110 1582 1525 470 176 181 54 100 100 0 1544 1432 444 266 286 94 100 100 0 1.1 1.157 1.158 1.191 1.366 1.148 0.91 80 484 2684 2515 0.138 1406 1278 1349 1166 0 0.08235
Hs6-1-19-1 4650 18600 110 3069 2955 770 172 177 51 100 100 0 4857 4484 1220 254 265 66 100 100 0 0.629 0.658 0.65 0.665 1.223 0.661 0.959 80 507 7500 7013 -0.668 2897 4603 2783 4230 0 -0.7363
Hs6-1-20-1 4830 18610 120 4981 4682 1793 169 176 54 100 100 0 3123 2920 1090 253 263 71 100 99 0 1.677 1.692 1.662 1.747 1.304 1.689 0.968 120 531 7682 7180 0.746 4812 2870 4513 2667 0 0.3666

Total number of rows: 20000

Table truncated, full table size 3721 Kbytes.




Supplementary file Size Download File type/resource
GSM141384.gpr.gz 1.9 Mb (ftp)(http) GPR
Processed data included within Sample table

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