normalization data transformation protocol
Gene level normalized expression signals were calculated from Affymetrix CEL files using RMA (Irizarry et al., 2003) for each experiment separately. ID_REF = VALUE = Log2 RMA-normalized expression signal
array scanning protocol
Scanning was done on an Affymetrix GeneChip Scanner 7G.
Affymetrix microarrays 430 2.0 arrays were hybridized with 5 micrograms of labeled, amplified cDNA , washed, stained and scanned according to the protocol described in Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol FS450_0001).
The tissue from six and three male animals was pooled for the analysis at P3 and at P14, respectively. For all samples described, 20 ng of total RNA was used and were amplified using a WT ovation Pico RNA amplification (Nugen, CA, USA).
nucleic acid extraction protocol
Total RNA from the different mouse brainstem regions were extracted using RNeasy mini kit (Qiagen). All RNA samples were DNase-treated and quantified on a NanoDrop spectrophotometer.
sample treatment protocol
Tissues were dissected in an ice cold, oxygenated bicarbonate buffered solution.