|
| Status |
Public on Mar 31, 2008 |
| Title |
MN, biological rep 5 |
| Sample type |
RNA |
| |
|
| Source name |
Ca. 100 MNs from neonatal spinal cord
|
| Organism |
Rattus norvegicus |
| Characteristics |
Wistar rats, age P0-P2
|
| Treatment protocol |
The spinal cords were disected out from neonatal animals, the tracer applied to cut axons to retrogradely label neurons through a ca. 3 hour incubation time in oxygenated normal calcium ringer at room temperature.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Spinal cords were snap frozen using CO2, cryosectioned into 10 micro meter slices, which were mounted onto POL-membrane metal frame slides (Leica) and fluorescently identied neurons were laser microdissected into a collector tube. Total RNA was extrated using the Picopure RNA isolation kit (Arcturus), which were then subject to a two round amplification using the RiboAmp HS RNA amplification kit (Arcturus) according to manufactors instructions. A DNAse treatment was included in the isolation protocol.
|
| Label |
biotin
|
| Label protocol |
Biotin labelled aRNA was generated from cDNA of the two-round amplification procedure using the GeneChip® Expression 3´-Amplification Reagents (Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 5 ug of aRNA were hybridized for 16 hr at 45 °C on GeneChip Rat Neurobiology U34. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data of fluorescently labelled neurons laser microdissected from 10 micro meter thick cryo-sections.
|
| Data processing |
Expression values were calculated using R and Bioconductor using the affy package. Li-wong expression summaries were calculated based on PM intensity values that were normalised (linear quantile plus quantile projection) and background subtracted (global minimization) (Ryge et al, 2007). The genes were filtered prior to analysis such that only probe sets annotated in the Ensembl database was included (using biomaRt bioconductor package), so the probe set was reduced from 1322 to 1050.
|
| |
|
| Submission date |
Oct 26, 2007 |
| Last update date |
Jan 09, 2014 |
| Contact name |
Jesper Ryge |
| E-mail(s) |
jesper.ryge@epfl.ch
|
| Phone |
+46707146879
|
| Organization name |
karolinska institutet
|
| Department |
neuroscience
|
| Lab |
mammalian locomotor lab
|
| Street address |
retziusvag 8
|
| City |
stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL88 |
| Series (1) |
| GSE9439 |
Gene expression profiles of two distinct neuronal populations in the neonatal rat spinal cord |
|
