|
| Status |
Public on Mar 03, 2014 |
| Title |
mouse cerebellar granule cells knockout 2 |
| Sample type |
RNA |
| |
|
| Source name |
CSTB KO_cerebellar granule cells at P5+2 DIV
|
| Organism |
Mus musculus |
| Characteristics |
strain background: 129SvJ
genotype/variation: cystatin B (CSTB)-deficient (Cstb-/-)
gender: female
developmental stage: P5+2 DIV
tissue: cerebellar granule cells
|
| Treatment protocol |
Mice were anesthetized with CO2 and sacrificed by decapitation. The dissected cerebella were homogenized using Lysing Matrix D tubes and FastPrep® FP120 Instrument with Proteinase K digestion (20 mg/ml, >30U/mg) according to the manufacturer’s instructions.
|
| Growth protocol |
Cstb-/- and wild type mice were grown under standard conditions in animal facilities. Cerebella from P5 pups were dissected and meninges removed in HHGN (1x HBSS, 2.5 mM HEPES, 35 mM glucose, 4 mM NaCO3). Intact cerebella were trypsinated in 10 mg/ml trypsin, 200 U/ml DNase in HHGN for 20 min at RT and triturated in 200 U/ml DNase in BME (Basal Medium Eagle). Cells were cultured in BME, 10% calf serum, 25 mM HCl. After 16-20 h from plating, 10 μM AraC (cytosine 1-β-d-arabinofuranoside) was added to prevent glial proliferation. Cells were harvested at DIV2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the ABI Prism 6100 Nucleic Acid PrepStation and PerfectPure RNA Cultured Cell kit according to the manufacturer’s instructions from cerebella and CGCs, respectively.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA.
|
| |
|
| Hybridization protocol |
The sample preparation and hybridzation of each cRNAs on Affymetrix GeneChip® Mouse Genome 430 2.0 arrays based on manufacturer's instruction.
|
| Scan protocol |
The quality control of each microarray was inspected using the Affymetrix GeneChip® Operating Software (GCOS) v1.4.
|
| Description |
CGC_KO2.CEL
Gene expression data from pre-symptomatic Cstb-/- mouse cerebellar granule cells.
|
| Data processing |
The raw data were imported to GeneSpring GX software and expression signal values were generated using the RMA (Robust Multiarray Average) algorithm (Irizarry et al., 2003) for background adjustment, quantile-normalization and summarization. Filtering of the probe sets was based on the expression with lower and upper cutoff 20 and 100 percentile, and on the fold-change (FC) with cutoff 1.3. In overall statistics of differentially expressed genes, t-test unpaired equal variance with corrected p-value ≤ 0.05 (cerebella) and p-value ≤ 0.01 (CGCs), with Benjamini and Hochberg multiple testing correction was used.
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| |
|
| Submission date |
May 30, 2013 |
| Last update date |
Mar 03, 2014 |
| Contact name |
Tarja Hannele Joensuu |
| E-mail(s) |
tarja.joensuu@helsinki.fi
|
| Phone |
+358-9-19125081
|
| Fax |
+358-9-19125073
|
| Organization name |
Folkhälsan Research Center
|
| Department |
Folkhälsan Institute of Genetics
|
| Street address |
Haartmaninkatu 8 (PO Box 63)
|
| City |
Helsinki |
| ZIP/Postal code |
000290 |
| Country |
Finland |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE47516 |
Gene expression alterations in the cerebellum and granule neurons of Cstb-/- mouse are associated with early synaptic changes and inflammation |
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