|
| Status |
Public on Oct 22, 2009 |
| Title |
AML332_6h_SB |
| Sample type |
RNA |
| |
|
| Source name |
AML PBMNC 6h SB stimulation
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: peripheral blood cells
disease status: Acute myeloid leukemia (AML)
treatment protocol: MPL receptor agonist (SB559457) stimulation
|
| Treatment protocol |
Cells were treated either with 5uM SB559457 of 2.8uM rhTpo for 6h prior to RNA isolation.
|
| Growth protocol |
Cells were grown in suspension culture (37C, 100% in humidity, 5% CO2) using Endothelial Cell Medium-2 (EGM-2) + 2%FBS (Cambrex, East Rutherford, NJ).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kits according to the manufacturer's recommendation.
|
| Label |
biotin
|
| Label protocol |
Microarray analysis was performed at the Cancer Center Microarray Facility at the University of Pennsylvania in accordance to their standard protocol. Briefly, samples were amplified with the NuGen WT Pico kit and labeled with the FL-Ovation cDNA Biotin Module V2 from NuGen. NuGen's fragmentation and labeling process combines enzymatic and chemical processes for fragmentation and amplified single-stranded cDNA to generate labeled targets suitable for Affymetrix GeneChip arrays. The first step is a combined chemical and enzymatic fragmentation that yields cDNA products ranging from 50-100 bases. In a second step, this fragmented product is labeled via enzymatic attachment of biotin-labeled nucleotide to the 3-hydroxyl end of the fragmented cDNA.
|
| |
|
| Hybridization protocol |
Hybridization to U133Av2 microarrays was done at 45ºC for 16 h . The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
Standard Affymetrix protocol was used (Cancer Center Microarray Facility at the University of Pennsylvania).
|
| Description |
PPG(2118) 332S U133Av2_GC-RMA
|
| Data processing |
Affymetrix Cel files were processed with Stratagene's ArrayAssist to yield chp files containing unlogged GC-RMA expression values and MAS5 detection flags.
|
| |
|
| Submission date |
Oct 21, 2009 |
| Last update date |
Oct 26, 2009 |
| Contact name |
Anna Kalota |
| E-mail(s) |
akalota@mail.med.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Medicine, Hem/Onc
|
| Street address |
421 Curie Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE18673 |
TpoR Agonist signaling in AML primary cells |
|
