|
| Status |
Public on Jun 12, 2014 |
| Title |
LNCaP-shFOXO4-2 |
| Sample type |
RNA |
| |
|
| Source name |
LNCaP cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: human LNCAP cell line
constuct: shFOXO4
|
| Treatment protocol |
LNCaP cells were infected with lentiviruses (pGIPZ-GFP-FOXO4 shRNA or pGIPZ control shRNA) and shortly selected with puromycin. To generate the spontaneous metastasis model, these cell lines (2 X 105) will be orthotopically injected into the prostates of male athymic nude mice (Taconic Inc., Germantown, NY) in a total volume of 50 µl of sterile Matrigel using a 30-gauge needle.
|
| Growth protocol |
Prostate cancer cell lines LNCaP were cultured in RPMI 1640 media supplemented with 10% FBS and incubated at 37ºC in a humidified incubator containing 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from six samples: LNCaP shFOXO4 cell line, LNCaP pGIPZ control cell line, LNCaP shFOXO4 primary tumor, LNCaP pGIPZ control primary tumor, LNCaP shFOXO4 lymph node metastases and LNCaP pGIPZ control lymph node metastases. Total RNA from frozen cell pellets and tissues was prepared using Trizol (Invitrogen) following manufacturer’s instructions. Before labeling, RNA samples were quantitated using a ND-1000 spectrophotometer (NanoDrop) and evaluated for degradation using a 2100 Bioanalyzer (Agilent Technologies). Samples are required to have a RIN >7, an OD 260:280 of 1.9-2.0, and an OD 260/230 >1.8 for gene expression array analysis. Expression profiling was accomplished using the Human HT-12 whole-genome gene expression array and direct hybridization assay (Illumina, Inc.).
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA was labeled using the Illumina Total Prep Amplification Kit (Life Technologies, Cat.# AMIL1791) following manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
750 ng of labeled cRNA was hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina, Cat# BD-103-0204) following manufacturer’s instructions
|
| Scan protocol |
Hybridized Beadchips were then scanned using the Illumina Iscan system
|
| Description |
LNCaP cellline, androgen dependent, shFOXO4 expressing
|
| Data processing |
Expression data are normalized using quantile normalization and differential expressed genes are identified using limma R package
|
| |
|
| Submission date |
Jun 11, 2014 |
| Last update date |
Jun 12, 2014 |
| Contact name |
Jianmin Wang |
| E-mail(s) |
Jianmin.Wang@roswellpark.org
|
| Organization name |
Roswell Park Comprehensive Cancer Center
|
| Department |
Bioinformatics and Biostatistics
|
| Street address |
Elm and Carlton Streets
|
| City |
Buffalo |
| State/province |
NY |
| ZIP/Postal code |
14263 |
| Country |
USA |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE58403 |
FOXO4 knockdown in LNCaP prostate cancer cells |
|
