|
| Status |
Public on Feb 16, 2016 |
| Title |
08-006H1_LN CRPC metastasis |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HGSv5.1.2
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
tissue: reference pool of LNCaP, DU145, PC3, and CWR22 cell lines
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To provide a reference standard RNA for use on two-color oligo microarrays, we pooled equal amounts of total RNA isolated from LNCaP, DU145, PC3, and CWR22 cell lines (American Type Culture Collection, Manassas, VA) growing at log phase in dye-free RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Reference RNA was purified using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Reference RNA was then further purified by RNeasy maxi kit and treated with DNAse using the RNase-Free DNase Set (Qiagen Inc, Valencia, CA) followed by two rounds of mRNA amplification with amino-allyl incorporation in the second round using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), according to the manufacturer’s specifications.
|
| Label |
Cy5
|
| Label protocol |
amplified amino-allyl aRNA from each experimental sample was labeled with Cy3 fluorescent dye (reference amino-allyl aRNA was labeled with Cy5) following the manufacturer’s suggested protocols.
|
| |
|
| Channel 2 |
| Source name |
08-006H1_LN
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
tissue: Samples were obtained from patients who died of metastatic CRPC and who signed written informed consent for a rapid autopsy performed within 6 hours of death, under the aegis of the Prostate Cancer Donor Program at the University of Washington. The Institutional Review Boards of the University of Washington and of the Fred Hutchinson Cancer Research Center approved this study. Visceral metastases were identified at the gross level, bone biopsies were obtained according to a template from 20 different sites and metastases identified at a histological level.
batch: 2
ne group: CHGA negative group
erg group: ERG positive group
tumor type: CRPC metastasis
tumor site: LN
sample_id: 08-006H1_LN
patient: 08-006
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eight-micron thick sections from soft tissue CRPC frozen metastases in Optimal Cutting Temperature compound (OCT; Tissue-Tek) were cut using a Leica CM3050S cryostat, collected on PEN Membrane Frame Slides (Life Technologies) and immediately fixed in 95% ethanol. Sections were briefly stained with hematoxylin then dehydrated in 100% ethanol. 5,000-10,000 tumor cells per sample were laser capture microdissected with an Arcturus Veritas instrument and collected on CapSure® Macro LCM Caps (Life Technologies). Digital photographs were taken of tissue sections before, during, and after LCM and assessed by a pathologist to confirm the tumor content. RNA was isolated using the Arcturus PicoPure RNA Isolation Kit and the samples were DNAse treated using the Qiagen RNase-Free DNase Set. Total RNA was amplified two rounds with amino-allyl incorporation using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
amplified amino-allyl aRNA from each experimental sample was labeled with Cy3 fluorescent dye (reference amino-allyl aRNA was labeled with Cy5) following the manufacturer’s suggested protocols.
|
| |
|
| |
| Hybridization protocol |
Hybridizations to Agilent 44K whole human genome expression oligonucleotide microarray slides (Agilent Technologies, Inc., Santa Clara, CA) following the manufacturer’s suggested protocols.
|
| Scan protocol |
Fluorescence array images were collected for Cy3 and Cy5 using the Agilent DNA microarray scanner G2505C (Agilent Technologies, Inc., Santa Clara, CA).
|
| Description |
08-006H1 CRPC lymph node metastasis
|
| Data processing |
Agilent Feature Extraction software versions 10.7.3.1 were used to grid and extract the data using the GE2_105_Jan09 protocols with default settings. Data was loess normalized within arrays (normexp background correction with offset 50) and quantile normalized between arrays in R using the Limma Bioconductor package by batch. Control probes were removed, duplicate probes averaged, and spots flagged by Agilent Feature Extraction software as being foreground feature nonuniformity or population outliers were assigned a value of NA. Data was normalized separately for LuCaP xenografts and CRPC metastases. CRPC metastases were subject to an additional normalization step to remove systematic batch effects due to date of RNA-amplification by application of the ComBat function within the sva Bioconductor package to the log2 Cy3 signal intensities. Batches are indicated in the ch2: characteristics: batch field and indicates RNA-amplification batch. Batch normalization was applied to a larger set of CRPC samples than those that appear in this study.
|
| |
|
| Submission date |
Feb 23, 2015 |
| Last update date |
Feb 16, 2016 |
| Contact name |
Ilsa Coleman |
| E-mail(s) |
[email protected]
|
| Phone |
206-667-1703
|
| Organization name |
Fred Hutchinson Cancer Center
|
| Department |
Human Biology
|
| Lab |
Peter Nelson
|
| Street address |
1100 Fairview Ave N, E2-112
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL15659 |
| Series (4)
|
| GSE66187 |
SRRM4 Expression and the Loss of REST Activity May Promote the Emergence of the Neuroendocrine Phenotype in Castration-Resistant Prostate Cancer |
| GSE74367 |
Characterizing the molecular features of ERG-positive tumors in primary and castration resistant prostate cancer |
| GSE74685 |
Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases |
| GSE77930 |
Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer |
|
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