|
| Status |
Public on Dec 01, 2012 |
| Title |
Spinal cord_ 15Day after injury_ replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Spinal cord, 15Day after injury
|
| Organism |
Danio rerio |
| Characteristics |
tissue: Spinal cord
age: 6 months adult
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the uninjured and injured spinal cords of the experimental fishes using Trizol (Invitrogen,15596018) and quality of RNA was checked on Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Assay). RNA was prepared from a minimum of 3 biological replicates. For each biological replicate, spinal cord from about 50-60 fishes were pooled in the control and also in the regenerating samples.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the Quick-Amp Labeling Kit, one-color kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following manufacturer instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent G2514F-024129 Agilent Custom Microarray GE 4x44K (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um).
|
| Description |
15Day-2
Gene expression in 15 day post injured spinal cord
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 021643_D_F_20081005) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jul 12, 2012 |
| Last update date |
Dec 01, 2012 |
| Contact name |
Justin Jeyakani Joseph Gnanakkan |
| E-mail(s) |
gnanakkan@gis.a-star.edu.sg
|
| Phone |
68088173
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Computational and Systems Biology
|
| Street address |
60, Biopolis street
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL15799 |
| Series (1) |
| GSE39295 |
Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish |
|
