|
| Status |
Public on Oct 17, 2006 |
| Title |
TTP_WT_120ActD_BioRep2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA prepared from fibroblast cell lines treated with 10% serum for 90 min followed by 0.5 µg/ml actinomycin D for 120 minutes
|
| Organism |
Mus musculus |
| Characteristics |
Cell line: Fibroblast TTP WT cell line prepared from littermate E14.5 mouse embryos, derived from parental TTP +/- male and TTP +/- female strain 129/B6; Embryo age: E14.5, Tissue: Whole embryo excluding head and organs
|
| Biomaterial provider |
Mice and fibroblast derived from mice were generated in the Perry J. Blackshear lab
|
| Treatment protocol |
Cells were grown in 100 mm dishes in 10% FBS/DMEM to approximately 70-80 % confluence, and were washed once in serum-free DMEM, and then incubated for 16 h in DMEM containing 0.5 % (v/v) FBS. The cells were then stimulated with 10% (v/v) FBS for 90 min then actinomycin D was added to a final concentration of 5 mg/ml for 120 minutes and were extracted for RNA.
|
| Growth protocol |
The fibroblasts were maintained at 37oC (5% CO2) in Dulbecco Minimal Essential Medium supplemented with 10% FBS, 2 mM L-glutamine, 100U/ml penicillin and 100 mg/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell monolayer was rinsed twice in cold PBS, 5 ml each. Each plate of cells was then lysed in 0.6 ml of Buffer RLT (RNeasy Mini Kit, Qiagen) and total cellular RNA was prepared following the RNeasy protocol.
|
| Label |
biotin
|
| Label protocol |
Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
15ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol.
|
| Scan protocol |
Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000. Data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
|
| Description |
Gene expression analysis was conducted using Mouse Genome 2.0 array (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol. For each array, 15ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000. Data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
|
| Data processing |
quantile normalization of CEL file, PDNN (PerfectMatch) estimation, log2 transformation
|
| |
|
| Submission date |
Jul 13, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
microarray@niehs.nih.gov, liuliw@niehs.nih.gov
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE5324 |
TTP mRNA targets identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts |
|
| Relations |
| Reanalyzed by |
GSE119085 |
