|
| Status |
Public on Jun 10, 2008 |
| Title |
22RV1 spink1 sirna rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
22RV1-non-targeting-siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
22RV1 prostate cancer cell lines transiently transfected with non targeting siRNA
|
| Treatment protocol |
Cells were not treated prior to RNA extraction
|
| Growth protocol |
22RV1 cells transfected with non-targeting siRNA or siRNA targeting SPINK1 were maintained in 10% RPMI media
|
| 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 |
22RV1-SPINK1-siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
22RV1 prostate cancer cell lines transiently transfected with siRNA targeting SPINK1
|
| Treatment protocol |
Cells were not treated prior to RNA extraction
|
| Growth protocol |
22RV1 cells transfected with non-targeting siRNA or siRNA targeting SPINK1 were maintained in 10% RPMI media
|
| 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 |
22rv1spink1/nontarget_dup
|
| 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. Over and under-expressed signatures were generated by filtering to include only features with significant differential expression (PValueLogRatio < 0.01) in all hybridizations and Cy5/Cy3 ratios > or < 1 (non-logged) in all hybridizations after dye flip correction
|
| |
|
| Submission date |
Apr 10, 2008 |
| Last update date |
Jun 10, 2008 |
| 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) |
| GSE11132 |
22RV1 prostate cancer cell line transfected with SPINK1 siRNA vs control siRNA |
|
