|
| Status |
Public on Dec 01, 2014 |
| Title |
Nr 3_AX-S |
| Sample type |
RNA |
| |
|
| Source name |
axonal compartment from cultured Smn knockdown motoneurons
|
| Organism |
Mus musculus |
| Characteristics |
cell type: motoneurons
subcellular compartment: axonal
genotype/variation: Smn knockdown
|
| Treatment protocol |
Motoneurons were treated with lentiviruses expressing an shRNA against Smn, empty vector control or left untreated (wild-type).
|
| Growth protocol |
Spinal cords were dissected from E12.5 mouse embryos and, following removal of dorsal root ganglia and meninges, motoneurons were isolated and enriched via p75-panning as described previously (Wiese et al. 2010). Following lentiviral transfection, one million motoneurons were plated into one compartment of a microfluidic chamber (Xona Microfluidics, SND 150) precoated with polyornithine and laminin-111 (Invitrogen, 23017–015). To achieve a directed growth of the axons through the microchannels of the microfluidic chamber towards the axonal compartment a BDNF gradient was established by adding this neurotrophic factor at 20 ng/ml only to the axonal compartment. CNTF (5 ng/ml) was applied to both compartments for survival. Motoneurons were grown for 7 days at 37°C and 5% CO2 in neurobasal medium (Invitrogen) containing 500 µM GlutaMAX (Invitrogen), 2% horse serum (Linaris), 2% B27 supplement (Invitrogen). 50% of culture medium was exchanged on day 1 and then every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of the somatodendritic and axonal compartment was extracted with the Pico Pure RNA Isolation Kit (Arcturus, KIT0204), according to the manufacturer´s protocol. Extracted RNA was directly used for linear amplification with the RiboAmp® HSPlus Amplification Kit (Arcturus, KIT0525). Linear amplification was done according to the manufacturer´s protocol with labelling of antisense RNA after two rounds of amplification. The biotin-labeled antisense RNA was then hybridised to a microarray chip (Affymetrix Gene Chip ® Mouse Genome 430 2.0 Array, 900496).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA (IVT-Express Kit, Technical Manual, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5 ug of cRNA were hybridized for 16 hr at 45C on a Mouse Genome 430 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station_450
|
| Scan protocol |
GeneChips were scanned using the GeneArray Scanner 3000
|
| Description |
Gene expression data from axonal compartment from cultured Smn knockdown motoneurons
|
| Data processing |
The data were analyzed with Bioconductor packages (under R) and quantile-quantile as normalization method
|
| |
|
| Submission date |
Jul 17, 2014 |
| Last update date |
Dec 01, 2014 |
| Contact name |
Susanne Elma Kneitz |
| E-mail(s) |
[email protected]
|
| Phone |
+49-931-31 86526
|
| Organization name |
University of Wuerzburg
|
| Department |
Physiologigcal Chemistry
|
| Street address |
Biozentrum, Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE59506 |
Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation |
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