|
| Status |
Public on Oct 01, 2013 |
| Title |
A549-pBABE_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Lung cancer cell line A549 with empty vector pBABE
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Non-small cell lung cancer
cell line: A549
gene transduction: pBABE empty vector
|
| Treatment protocol |
Cell lines were stably transduced with pBABE retroviral constructs expressing wild-type LKB1 (LKB1-WT), or controls of empty pBABE (pBABE) or kinase dead mutant LKB1 (LKB1-K78I). Selection for cell lines with stable expression of these constructs was achieved by culturing cells in growth media containing 1.0 micrograms/milliliter puromycin for two weeks.
|
| Growth protocol |
Cell lines were grown in RPMI1640 containing 5% fetal bovine serum, without antibiotics. Cells were harvested during logarithmic growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. Isolated RNA was bioanalyzed using Agilent to determine 260/280 ratio, RNA concentration, and RIN.
|
| Label |
Biotin
|
| Label protocol |
Ambion WT Expression reactions for all samples were started using 130ng of total RNA. Resulting cRNA products from the overnight IVT reactions were cleaned with Ambion-WT bead cleanup kit. 10.5ug cRNA was used for cDNA synthesis. Resulting cDNA was cleaned with Ambion-WT bead cleanup kit. 5.5ug of cDNA was used for biotin labeling reactions.
|
| |
|
| Hybridization protocol |
5.5ug of fragmented, biotinylated cDNA were hybridized for 16 hours to a HT Human Gene 1.1 ST PM16 array plate utilizing the GeneTitan instrument.
|
| Scan protocol |
HT Human Gene 1.1 ST chips were scanned using scanned on the Affymetrix Gene Titan AGCC v. 3.2.3
|
| Description |
A549 cells stably expressing pBABE empty vector
|
| Data processing |
CEL files were analyzed on Affymetrix Expression Console v. 1.1 using a RMA normalization algorithm producing log base 2 results.
|
| |
|
| Submission date |
Sep 30, 2013 |
| Last update date |
Oct 01, 2013 |
| Contact name |
David Carbone |
| E-mail(s) |
david.carbone@osumc.edu
|
| Organization name |
Ohio State University
|
| Department |
Internal Medicine
|
| Street address |
460 West 12th Avenue; 488 BRT
|
| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL11532 |
| Series (1) |
| GSE51266 |
Gene expression changes induced by LKB1 expression in human lung adenocarcinoma cell lines |
|
