|
| Status |
Public on Jul 28, 2011 |
| Title |
LNCaP Cell Line, Control siRNA versus PCAT1-siRNA-4, rep 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LNCaP prostate cancer stem cells, PCAT1 siRNA-4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
cell type: prostate cancer
treatment: siRNA-PCAT1-4
|
| Treatment protocol |
Cells were plated in 100mM plates at a desired concentration and transfected with 20uM experimental siRNA oligos or non-targeting controls twice, at 12 hours and 36 hours post-plating. Knockdowns were performed with Oligofectamine and Optimem. Knockdown efficiency was determined by qPCR.
|
| Growth protocol |
Cells were grown in RPMI 1640 (Invitrogen) and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol and an RNeasy Kit (Invitrogen) with DNase I digestion according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label.
|
| |
|
| Channel 2 |
| Source name |
LNCaP prostate cancer stem cells, control siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
tissue: prostate cancer
treatment: siRNA-control
|
| Treatment protocol |
Cells were plated in 100mM plates at a desired concentration and transfected with 20uM experimental siRNA oligos or non-targeting controls twice, at 12 hours and 36 hours post-plating. Knockdowns were performed with Oligofectamine and Optimem. Knockdown efficiency was determined by qPCR.
|
| Growth protocol |
Cells were grown in RPMI 1640 (Invitrogen) and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol and an RNeasy Kit (Invitrogen) with DNase I digestion according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
Biological replicate 1 of 3.
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Jun 10, 2011 |
| Last update date |
Jul 28, 2011 |
| Contact name |
Matthew Iyer |
| E-mail(s) |
mkiyer@med.umich.edu
|
| Phone |
7346154503
|
| Organization name |
University of Michigan
|
| Department |
Michigan Center for Translational Pathology
|
| Lab |
Chinnaiyan Lab
|
| Street address |
5316 CCGC 0940 1400 E. Medical Center Drive
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-0940 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (2) |
| GSE29886 |
LNCaP Prostate Cancer Cells: Control vs. siRNA-PCAT1 Treated |
| GSE31728 |
Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression |
|
