|
| Status |
Public on Oct 15, 2012 |
| Title |
MM line transfected with shERK5, rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Stable transfection with shERK5 SureSilencing plasmid
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMESO
cell type: epithelioid malignant mesothelioma (MM)
transfection: shERK5
property: ERK5>70% inhibited
|
| Treatment protocol |
In order to achieve inhibition of ERK5, mesothelioma cells (HMESO) were stably transfected with shERK5 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 ERK5.
|
| 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 RNAs 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 that were hybridized to the probe array at 45 oC 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 ERK5 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 RMA method. Data were log transformed.
|
| |
|
| Submission date |
Oct 15, 2012 |
| Last update date |
Oct 15, 2012 |
| 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 |
|
