tissue: brain treatment: spinal cord transection sample type: did not regenerate their axons
Extracted molecule
total RNA
Extraction protocol
The total RNA isolated using miRNeasy Micro Kit (cat.no. 217084, Qiagen Germantown, MD). The total RNA was then amplified using Ovation® Pico WTA System V2 (cat.no. 3302-12, NuGEN Technologies, Inc., San Carlos, CA)
Label
biotin
Label protocol
The amplified SPIA cDNA (5µg) was labeled using Encore Biotin Module V2 (cat.no. 4200-12, NuGEN Technologies, Inc., San Carlos, CA).
Hybridization protocol
After labeling, 25 µg of cDNA were hybridized for 18 hours at 45°C to Genechip Zebrafish Genome Array. Genechips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix Genechip scanner 3000 with 7G upgrade
Description
gene expression from cerebrospinal neurons that did not regenerate their axons
Data processing
The .cel files were obtained with Affymetrix® GeneChip® Command Console® Software (AGCC). Data was analyzed in GeneSpring GX 12.6.1 (Agilent Technologies, Santa Clara, CA). Robust Multi-array Average (RMA) (Irizarry, 2003.) summarization algorithm (with Quantile Normalization and Median Polish probe summarization procedure) and baseline transformation (baseline to median of all 9 samples) was run on data using logarithmic scale. Probe sets were filtered by expression values based on percentiles (20th to 100th). For comparison of the 3 groups, ANOVA with Tukey HSD post-hoc test was done to identify statistically significant (p-value cutoff 0.05) differentially expressed transcripts. Statistical test p-values were corrected for False Discovery Rate (FDR) by Benjamini-Hochberg (BH) procedure. Differentially expressed transcripts were annotated with GeneSpring GX 12.6.1.
