|
| Status |
Public on Aug 07, 2007 |
| Title |
RWPE-ETV1_dye_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RWPE-ETV1
|
| Organism |
Homo sapiens |
| Characteristics |
benign prostate cell line RWPE stably expressing ETV1
|
| Treatment protocol |
Cells were not treated prior to RNA extraction.
|
| Growth protocol |
RWPE-GUS or RWPE-ETV1 cells were maintained in Keratinocyte-SFM supplemented with bovine pituitary extract (25 mg/ 500 ml) human recomninant EGF (2.5 ug/500 ml medium) and 3ug/ml blasticidin
|
| 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 |
RWPE-GUS
|
| Organism |
Homo sapiens |
| Characteristics |
benign prostate cell line RWPE stably expressing the GUS control gene
|
| Treatment protocol |
Cells were not treated prior to RNA extraction.
|
| Growth protocol |
RWPE-GUS or RWPE-ETV1 cells were maintained in Keratinocyte-SFM supplemented with bovine pituitary extract (25 mg/ 500 ml) human recomninant EGF (2.5 ug/500 ml medium) and 3ug/ml blasticidin
|
| 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 4x44k format two-color arrays.
|
| Description |
na
|
| 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. To identify differentially expressed features, we filtered the data to only include features showing significant differential expression in (PValueLogRatio < 0.01) in all four hybridizations with at least 2 fold average over- or under-expression (LogRatio) after dye flip correction.
|
| |
|
| Submission date |
May 03, 2007 |
| Last update date |
Aug 07, 2007 |
| Contact name |
Scott Tomlins |
| E-mail(s) |
tomlinss@med.umich.edu
|
| 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 |
GPL4133 |
| Series (1) |
| GSE7701 |
Profiling of RWPE cells stably over-expressing ETV1 |
|
