|
| Status |
Public on Dec 21, 2012 |
| Title |
p100(-/-)_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
p100(-/-)
|
| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6
development stage: E14.5
genotype/variation: NF-kB2/p100-deficient (p100-/-)
tissue origin: embryo
cell type: mouse embryonic fibroblasts (MEF)
|
| Treatment protocol |
At age passage 3, MEFs were left untreated and total RNA was isolated for microarray experiments. To paralelly monitor signaling events, cytoplasmic and nuclear proteins were extracted, as well.
|
| Growth protocol |
Mouse embryonic fibroblasts (MEFs) were isolated from E14.5 wild-type and p100-/- mouse embryos. Five independent wild-type and five independent p100-/- fibroblast cell lines were cultured at 37C in Dulbecco’s modified Eagle's medium (GIBCO/Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Cölbe, Germany), penicillin (100 U/ml), streptomycin (100 mg/ml) and L-glutamine (2 mM) (GIBCO/Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total cellular RNA was isolated using RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions, possible contamination by genomic DNA was removed by DNAseI treatment using the RNAse-Free DNAse Set (Qiagen), quality was checked by spectrophotometry and agarose gel electrophoresis, 2ug RNA per sample was used for cDNA preparation when A260:A280 ratio was 1.8-2.1 and RNA was intact
|
| Label |
biotin
|
| Label protocol |
according to manufacturers instructions (http://www.appliedmicroarrays.com/pdf/codelink_bioarray_processing_protocol.pdf)
|
| |
|
| Hybridization protocol |
according to manufacturers instructions (http://www.appliedmicroarrays.com/pdf/codelink_bioarray_processing_protocol.pdf)
|
| Scan protocol |
GenePix 4000B Array Scanner and GenePix Pro 4.0 software according to the settings suggested by GE Healthcare (supplier of CodeLink microarrays at experiment time, http://www.appliedmicroarrays.com/pdf/codelink_bioarray_processing_protocol.pdf)
|
| Data processing |
Raw data were analysed using Codelink Expression Analysis v.4.1 software to extract MDFCs (minimal detectable fold changes). Subsequent analysis was done using R and Bioconductor. Only probes with probe type DISCOVERY were considered (34967 transcripts). Intensity values <0.01 were set to 0.01. Raw intensities were scaled to array median. After logarithmizing, quantile normalization was applied across all arrays.
|
| |
|
| Submission date |
Jun 30, 2011 |
| Last update date |
Dec 21, 2012 |
| Contact name |
Daniela Albrecht |
| E-mail(s) |
daniela.albrecht@biocontrol-jena.com
|
| Organization name |
BioControl Jena GmbH
|
| Street address |
Wildenbruchstr. 15
|
| City |
Jena |
| ZIP/Postal code |
07745 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8063 |
| Series (1) |
| GSE30317 |
p100 deficiency alone is insufficient for full activation of the alternative NF-κB pathway: TNF signaling cooperates with p52-RelB activation in the transcriptional regulation of the enpp2/autotaxin promoter |
|
