|
| Status |
Public on May 23, 2012 |
| Title |
N22 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pooled benign prostate tissue (Clontech)
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
One ug of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| Channel 2 |
| Source name |
prostate tissue (radical prostatectomy specimen)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: benign prostate tissue (N)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
One ug of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| |
| Hybridization protocol |
825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65 C hybridization oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to the manufacturer's recommnded protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent). Arrays were washed according to the manufacturer's recommended protocol including the Stabilization and Drying Solution step (Two-Color Microarray-Based Gene Expression Analysis, Agilent).
|
| Scan protocol |
Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505B, Agilent) using the default settings for 1x44k or 4x44k format two-color arrays.
|
| Data processing |
Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software (Version 9.1.3.1). Spot values were normalized using the default linear-lowess normalization. LogRatio for all probes, which were used for subsequent analysis, were converted to log(2). Although the 1x44k and 4x44k arrays have the same probes, the 4x44k arrays have 10 replicates of some probes. Thus, to generate a final data set, the median value of replicated probes was used for 4x44k arrays. The final data set (including benign prostate, localized prostate cancer and CRPC) was uploaded into a custom instance of Oncomine for automated analysis. In Oncomine, the dataset was median centered (per array) prior to indicated analyses.
|
| |
|
| Submission date |
Feb 21, 2012 |
| Last update date |
May 23, 2012 |
| Contact name |
Scott Tomlins |
| E-mail(s) |
[email protected]
|
| 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 |
GPL6480 |
| Series (1) |
| GSE35988 |
The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer |
|
