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Sample GSM198176 Query DataSets for GSM198176
Status Public on Jul 31, 2007
Title STROMA_NOR_2
Sample type genomic
 
Channel 1
Source name STROMA_NOR_2
Organism Homo sapiens
Characteristics Normal Stroma - Organ Donor
Extracted molecule genomic DNA
Extraction protocol 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. Genomic DNA was isolated from the cells using QIAamp DNA Mini Kit (Qiagen, Valencia, CA) and DNA concentration was determined using Quant-iT DNA Assay Kit, High Sensitivity (Invitrogen, Carlsbad, CA).
Label Cy5
Label protocol For each sample, 100 ng of normal genomic DNA was converted into a WGA library in a volume of 15 ul and amplified by PCR following the manufacturer’s WGA amplification protocol. Hundred ng aliquots of the products were then subjected to a second WGA PCR amplification in the presence of amino-allyl dUTP and purified. Yields after all WGA PCR amplifications were between 10 to 20 μg per reaction and used for the hybridizations. For all WGA amplified genomic DNA samples with aminoallyl dUTP incorporated (4 μg), nuclease free water was added to 500 μl, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 12,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 12,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 μl with nuclease free water. For labeling, 5 μl of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 10 min. Two μl of anhydrous DMSO were added to Alexa Fluor 647 (Cy5) and 555 (Cy3) dye packs (Invitrogen, Carlsbad, CA), mixed thoroughly, and 2 μl of the appropriate 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 μl of 4 M Hydroxylamine and incubated in room temperature for 15 min. The Cy3 labeled and Cy5 labeled genomic DNA for each hybridization were mixed, and RNase free water was added to 100 μl, and purified using Qiaquick (Qiagen, Valencia, CA) spin columns. The final eluted volume was 60 μl.
 
Channel 2
Source name Human Genomic DNA : Male
Organism Homo sapiens
Characteristics Promega Normal Genomic DNA
Extracted molecule genomic DNA
Extraction protocol 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. Genomic DNA was isolated from the cells using QIAamp DNA Mini Kit (Qiagen, Valencia, CA) and DNA concentration was determined using Quant-iT DNA Assay Kit, High Sensitivity (Invitrogen, Carlsbad, CA).
Label Cy3
Label protocol For each sample, 100 ng of normal genomic DNA was converted into a WGA library in a volume of 15 ul and amplified by PCR following the manufacturer’s WGA amplification protocol. Hundred ng aliquots of the products were then subjected to a second WGA PCR amplification in the presence of amino-allyl dUTP and purified. Yields after all WGA PCR amplifications were between 10 to 20 μg per reaction and used for the hybridizations. For all WGA amplified genomic DNA samples with aminoallyl dUTP incorporated (4 μg), nuclease free water was added to 500 μl, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 12,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 12,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 μl with nuclease free water. For labeling, 5 μl of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 10 min. Two μl of anhydrous DMSO were added to Alexa Fluor 647 (Cy5) and 555 (Cy3) dye packs (Invitrogen, Carlsbad, CA), mixed thoroughly, and 2 μl of the appropriate 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 μl of 4 M Hydroxylamine and incubated in room temperature for 15 min. The Cy3 labeled and Cy5 labeled genomic DNA for each hybridization were mixed, and RNase free water was added to 100 μl, and purified using Qiaquick (Qiagen, Valencia, CA) spin columns. The final eluted volume was 60 μl.
 
 
Hybridization protocol For hybridization, 40 μg of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 60 μl of labeled genomic DNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 12,000 rcf for 8 min at RT. The spin column was inverted in a new collection tube and centrifuged at 12,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18 μl with nuclease free water and the following were added: 2 μl of yeast tRNA (10 μg/μl, Invitrogen, Carlsbad, CA), 4 μl of Poly dA-Poly dT (10 μg/μl, Sigma-Aldrich, St. Louis, MO), 5.1 μl of 20X SSC and 0.9 μl of 10% SDS. The probe was denatured at 100° C for 3 min and centrifuged at 12,000 rcf for 10 sec. The probe was added directly to the microarray and a cover slip was placed carefully. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65° C water bath. After hybridization, cover slips were removed by placing slides in 2x SSC / 0.03% SDS. Slides were then washed sequentially in the following order: 2x SSC / 0.03% SDS for 5 min, 1x SSC / 0.03% SDS for 5 min, and 0.2x SSC / 0.03% SDS for five minutes. Slides were given a final wash in 0.2x SSC for two minutes, 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 N_2005-02-23_NM_Num85_0001.gpr
Data processing Images were gridded and spots were quantified using GenePix Pro 6.0 software (Axon Instruments, Union City, CA).
 
Submission date Jun 05, 2007
Last update date Jun 05, 2007
Contact name Jung Kim
E-mail(s) junghk@med.umich.edu
Organization name University of Michigan
Street address 1400 E. Medical Center Drive
City Ann Arbor
State/province MI
ZIP/Postal code 48109
Country USA
 
Platform ID GPL2013
Series (1)
GSE8026 Integrative Analysis of Genomic Aberrations Associated with Prostate Cancer Progression

Data table header descriptions
ID_REF
VALUE The Median of Ratios (log2 of Cy5/Cy3) for each included feature was normalized using locally weighted regression (lowess) with a window of 0.6 using custom software written in Perl and R.

Data table
ID_REF VALUE
Hs6-13-18-25 -0.030833333
Hs6-30-20-10 -0.464
Hs6-8-8-2 -0.030833333
Hs6-21-12-17 -0.451333333
Hs6-17-6-2 -0.2198
Hs6-3-15-6 -0.4355
Hs6-4-21-3 -0.220666667
Hs6-5-21-15 -0.4795
Hs6-8-21-8 -0.156
Hs6-22-12-22 0.0205
Hs6-2-3-7 -0.210666667
Hs6-25-1-2 -0.418
Hs6-28-15-4 0.13525
Hs6-29-9-10 0.297333333
Hs6-32-19-3 0.0188
Hs6-4-13-6 -0.210666667
Hs6-7-13-4 0.129
Hs6-9-15-18 -0.0724
Hs6-10-22-12 -0.104
Hs6-1-3-24 -0.376833333

Total number of rows: 13653

Table truncated, full table size 284 Kbytes.




Supplementary file Size Download File type/resource
GSM198176.gpr.gz 1.9 Mb (ftp)(http) GPR
Processed data included within Sample table

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