Genomics

Dataset Information

23

Integrative Analysis of Genomic Aberrations Associated with Prostate Cancer Progression


ABSTRACT: Genome-wide copy number changes were monitored using array comparative genomic hybridization (aCGH) of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression including precursor lesions, clinically localized disease and metastatic disease. A total of 62 specific cell populations from 38 patients were profiled. Keywords: Disease state analysis using array-based comparatavie genomic hybridization Overall design: Experiment Design: - Type of experiment: This study used aCGH experiments to profile genome-wide copy number changes through the isolation of pure cell populations representing entire spectrum of prostate cancer disease using laser capture microdissection (LCM) and OmniPlex Whole Genomic (WGA) Amplification. - Experimental factors: This study profiled various pure cell populations isolated by LCM from prostate tissue. - The number of hybridizations performed in the experiment: A total of 62 hybridizations were performed. - The type of reference used for the hybridizations: A single commercially available pool of normal human male genomic DNAs (Promega Inc.) was used as the reference in all hybridizations. For the reference 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, purified, and used for the hybridizations. - Hybridization design: For all hybridizations, the experimental prostate sample isolated by LCM was labeled with Cy5 and the common reference of benign pooled prostate as described above was labeled with Cy3. - Quality control steps taken: We purchased human genomic DNA samples having multiple copies (1 to 5) of X chromosome from NIGMS Human Genetic Mutant cell repository (http://www.nigms.nih.gov/Initiatives/HGCR/) and amplified. The hybridization result obtained from original samples and WGA-amplified samples were cross-compared. Also, genomic DNA from both epithelium and stroma of normal prostate tissue from a cadaveric donor, previously diagnosed with Down’s syndrome, was hybridized for the detection of a single copy gain in chromosome 21. - The mRNA expression data is also available from GEO (GSE6099) and Oncomine (www.oncomine.org). Samples used, extract preparation and labeling: - The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: All samples used were of human origin. Tissues were from the radical prostatectomy series at the University of Michigan and from the Rapid Autopsy Program, which are both part of University of Michigan Prostate Cancer Specialized Program of Research Excellence Tissue Core. All samples were collected with informed consent of the patients and prior institutional review board approval. The table in the MIAME Checklist (Supplementary Methos) summarizes the samples used. For prostate cancer samples, the Gleason patterns present on the sample section used are indicated, as well as the Gleason patterns of the actual cells isolated by LCM. A unique case number is given. Descriptions of samples are given where applicable, including the location of metastatic foci. - Manipulation of biological samples and protocols used: 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. 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). - Protocol for preparing the hybridization extract: 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. - Labeling protocol(s): 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. - External controls (spikes): Not utilized Hybridization procedures and parameters: - The protocol and conditions used during hybridization, blocking and washing: 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. Measurement data and specifications: - Type of scanning hardware and software used: Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA). - Type of image analysis software used: Images were gridded and spots were quantified using GenePix Pro 6.0 software (Axon Instruments, Union City, CA). - A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis: GenePix Pro 6.0 are described in the sample files. For all hybridizations, the log2 normalized Median of Ratios (as described below) was used. - The complete output of the image analysis before data selection and transformation (spot quantitation matrices): The aCGH raw data files will be available from GEO. The mRNA expression data is already available from GEO (GSE6099) and Oncomine (www.oncomine.org) under the study name Tomlins_Prostate_Chinnaiyan-Lab_2. - Data selection and transformation procedures: Arrays were auto-gridded by GenePix 6.0. Features flagged by GenePix as not found during grid alignment and areas of obvious defects were manually flagged and both were excluded from further analysis. 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. To exclude unreliable features, features showing an average normalized Median of Ratios of > 1.5 or < 0.75 across a series of self-self hybridizations (including unamplified and WGA amplifi

INSTRUMENT(S): Chinnaiyan Human 20K Hs6

SUBMITTER: Jung Kim  

PROVIDER: GSE8026 | GEO | 2007-07-31

SECONDARY ACCESSION(S): PRJNA100833

REPOSITORIES: GEO

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Publications


Integrative analysis of genomic aberrations in the context of trancriptomic alterations will lead to a more comprehensive perspective on prostate cancer progression. Genome-wide copy number changes were monitored using array comparative genomic hybridization of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression, including precursor lesions, clinically localized disease, and metastatic disease. A total of 62 specific cell populations from 38 patien  ...[more]

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