|
| Status |
Public on May 08, 2010 |
| Title |
MM line transfected with shERK2, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Stable transfection with shERK2 SureSilencing plasmid
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMESO
cell type: epithelioid malignant mesothelioma (MM)
transfection: shERK2
property: ERK2>70% inhibited
|
| Treatment protocol |
In order to achieve inhibition of ERK1 or ERK2, mesothelioma cells (HMESO) were stably transfected with either shControl, shERK1 or shERK2 plasmid from SABiosciences using Lipofectamine 2000. After selection in G418 (400ug/ml) medium for 14 days, limited dilution was performed to get pure clones of more than 70% inhibited ERK1 or ERK2.
|
| Growth protocol |
All cells were incubated at 37oC and 5% CO2 and grown to approximately 80-90% confluency in DMEM/F12 medium at a 1:1 ratio containing 10% fetal bovine serum (FBS), 0.1 ug/ml hydrocortisone, 2.5 ug/ml insulin, 2.5 ug/ml transferrin and 2.5 ng/ml sodium selenite and penicillin-streptomycin (50 U/ml penicillin G, 50 ug/ml streptomycin sulfate).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using an RNeasy Plus Mini kit according to the manufacturer's protocol (Qiagen, Valencia, CA). RNA was dissolved in RNAse-free water and quantified using the NanoDrop spectrophotometer.
|
| Label |
Biotin
|
| Label protocol |
RNA was synthesized to ds cDNA which was then converted to amplified ss cDNA using a Nugen Ovation V2 system. ss cDNA was Biotinylated using TdT end label.
|
| |
|
| Hybridization protocol |
Biotin-labeled cDNA fragments were hybridized to the probe array at 45oC for 16 h.
|
| Scan protocol |
The probe array were scanned (Affymetrix GeneArray Scanner, GS3000) to quantify the fluorescence intensity associated with each oligonucleotide probe.
|
| Description |
Gene expression data in ERK2-inhibited epithelioid mesothelioma cell line.
|
| Data processing |
A human U133A 2.0 array was scanned twice, the images overlaid, and the average intensities of each probe cell compiled. Microarray data were analyzed using GeneSifter software (VizX Labs, Seattle, WA) by the RMA method. Data were log transformed.
|
| |
|
| Submission date |
May 07, 2010 |
| Last update date |
May 07, 2010 |
| Contact name |
Arti Shukla |
| E-mail(s) |
Arti.Shukla@uvm.edu
|
| Phone |
802-656-8253
|
| Organization name |
University of Vermont
|
| Department |
Pathology
|
| Lab |
215 HSRF
|
| Street address |
89 Beaumont Avenue
|
| City |
Burlington |
| State/province |
VT |
| ZIP/Postal code |
05405 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE21750 |
Expression data from mesothelial (LP9) and mesothelioma cells (HMESO) inhibited for extracellular-regulated kinase (ERK) 1, 2, or 5 |
|
