|
| Status |
Public on Oct 21, 2013 |
| Title |
Mill MM line transfected with shCREB1, biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Stable transfection with shCREB1 SureSilencing plasmid
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: fibrosarcomatoid mesothelioma cell line H2373/PPMMill
genotype/variation: CREB inhibition
|
| Treatment protocol |
In order to obtain CREB inhibition human malignant mesothelioma cells were transfected with SureSilencing CREB1 plasmid (SuperArray Biosciences) using Lipofectamine 2000. After selecting for 15 days in G418 (400ug/ml) containing medium, limited dilution was performed to obtain clones showing ≥70% inhibition.
|
| 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 CREB1 inhibited fibrosarcomatoid mesothelioma cell line
CREB1>70% inhibited
|
| 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 21, 2013 |
| Last update date |
Oct 21, 2013 |
| 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) |
| GSE51447 |
Microarray data from CREB inhibited and control mesothelioma cells |
|
