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.
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_2005-03-10_NM_Num46_0001.gpr
Data processing
Images were gridded and spots were quantified using GenePix Pro 6.0 software (Axon Instruments, Union City, CA).
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.
