|
| Status |
Public on Nov 01, 2009 |
| Title |
LAPC4_10nM |
| Sample type |
RNA |
| |
|
| Source name |
treated tissue culture cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human prostate cancer cells
cell line: LAPC4
|
| Treatment protocol |
24 hours after maintaining the cells in androgen-free media (media supplemented with 10 % charcoal-stripped serum (Hyclone)), cells were treated with 0, 0.1, 1 or 10 nM R1881 and total RNA isolated with Trizol reagent (InVitrogen).
|
| Growth protocol |
3.7 million LAPC-4 cells were seeded in 75-cm flasks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA isolated with Trizol reagent (InVitrogen).
|
| Label |
Cy3
|
| Label protocol |
Total RNA was labeled with Cy3 as described (Thomson et al., 2004)
|
| |
|
| Hybridization protocol |
10 micrograms of RNA was Cy3-labeled and hybridized to custum microarrays. The custom microarrays contained oligonucleotide probes complementary to 474 human miRNAs were synthesized by Combimatrix. Probes containing 2 mismatches were included for all miRNAs. Arrays were pre-hybridized at 37 degrees for 1 hour in 3X SSC, 0.1% SDS, 0.2% BSA. Labeled RNA was hybridized to arrays at 37 degrees overnight in 400mM Na2HPO4 (pH 7.0), 0.8% BSA, 5% SDS, and 12% formamide. Arrays were washed once at room temperature in 2X SSC, 0.25% SDS, 3 times at room temperature in 1.6X SSC, and twice in ice-cold 0.8X SSC.
|
| Scan protocol |
Hybridized arrays were scanned using a GenePix 4000B microarray scanner (Axon) and signal intensities were extracted using the Combimatrix Microarray Imager software.
|
| Description |
10 micrograms of RNA was Cy3-labeled and hybridized to custum microarrays. The custom microarrays contained oligonucleotide probes complementary to 474 human miRNAs were synthesized by Combimatrix
|
| Data processing |
The background value was determined by calculating the median signal from the mismatch probes and this value was subtracted from all perfect match probes. Signals that were less than 1.5 times background were removed and datasets were median centered prior to calculating fold-change values.
|
| |
|
| Submission date |
May 22, 2009 |
| Last update date |
May 22, 2009 |
| Contact name |
Shawn E Lupold |
| E-mail(s) |
[email protected]
|
| URL |
http://urology.jhu.edu/shawnlupold/index.php
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Urology
|
| Street address |
600 N Wolfe St
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL8573 |
| Series (2) |
| GSE16213 |
Androgen repsonsive microRNAs in LAPC-4 cell lines |
| GSE16225 |
Androgen repsonsive microRNAs |
|
