|
| Status |
Public on Feb 19, 2015 |
| Title |
cornea vs epidermis replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cornea
|
| Organism |
Danio rerio |
| Characteristics |
age: adult
tissue: cornea
|
| Treatment protocol |
To prepare total RNA from adult tissues of the cornea and the skin, AB2O2 wild type strain of adult zebrafish was deeply anaesthetised by lethal dose of MESAB. For each biological repeat of the microarray analysis, 30 globes were removed from the sockets and the cornea was carefully isolated with forceps from the other periocular tissues (the sclera, lens, iris, periocular muscles and retina) in the phosphate-buffered saline. Skin dermis was prepared from ten fish by scraping the scales that hold epidermis on their outer surface with a scalpel that was kept at the right angle moving from tail to head direction. Inevitable minimal amount of fin and muscle tissues that could not be separated was allowed to be included into the dermis preparation. Skin epidermis was prepared from scales as described above. Tissues were immediately chilled on crashed blocks of dry ice and weighted.
|
| Growth protocol |
Adult zebrafish from the fish facility were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from frozen tissues were extracted in Trizol with an electric homogenizer.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription reaction of the isolated total RNA followed by cRNA probe synthesis were conducted by using Low RNA Input Linear Amplification kit (Cat. No. 51843523, Agilent Technologies). Synthesized cRNA probes were purified by QIAgen RNeasy purification kit and quantified by NanoDrop ND-1000 UV-VIS Spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| Channel 2 |
| Source name |
epidermis
|
| Organism |
Danio rerio |
| Characteristics |
age: adult
tissue: epidermis
|
| Treatment protocol |
To prepare total RNA from adult tissues of the cornea and the skin, AB2O2 wild type strain of adult zebrafish was deeply anaesthetised by lethal dose of MESAB. For each biological repeat of the microarray analysis, 30 globes were removed from the sockets and the cornea was carefully isolated with forceps from the other periocular tissues (the sclera, lens, iris, periocular muscles and retina) in the phosphate-buffered saline. Skin dermis was prepared from ten fish by scraping the scales that hold epidermis on their outer surface with a scalpel that was kept at the right angle moving from tail to head direction. Inevitable minimal amount of fin and muscle tissues that could not be separated was allowed to be included into the dermis preparation. Skin epidermis was prepared from scales as described above. Tissues were immediately chilled on crashed blocks of dry ice and weighted.
|
| Growth protocol |
Adult zebrafish from the fish facility were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from frozen tissues were extracted in Trizol with an electric homogenizer.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription reaction of the isolated total RNA followed by cRNA probe synthesis were conducted by using Low RNA Input Linear Amplification kit (Cat. No. 51843523, Agilent Technologies). Synthesized cRNA probes were purified by QIAgen RNeasy purification kit and quantified by NanoDrop ND-1000 UV-VIS Spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| |
| Hybridization protocol |
Hybridisation of the microarray was done at 65oC for 17 hours, followed by stringency wash.
|
| Scan protocol |
Scanning was done with the Axon model 4000B dual-laser scanner and the corresponding GenePix6 software (Molecular Devices, Union City, CA). Two emission channels for Cy3 and Cy5 dyes were scanned in parallel (16-bit colour depth).
|
| Description |
77_block1.gpr
biological repeat 3
|
| Data processing |
The probe sequences commonly found on the Agilent G2518A (design ID:013223 Option 001, 60-mer oligos) and G2519F microarray (design ID: 019161-V2) were re-mapped onto the Zebrafish genome Zv8 and Unigene ESTs by an in-house Perl script after either BLAT- or BLAST-sequence alignment (Altschul, Madden et al. 1997; Kent 2002). 16450 out of 23763 Agilent probes (69.2%) can be re-mapped onto databases of either Unigene (zebrafish build #117), Ensembl genes (Zv8, release 54) or RefSeq genes (zebrafish release 38). BLAT-/BLAST hits within the coding region or within 500 bp range of either 5'- or 3'-flanking region of start or stop codon, respectively, were regarded as the hit (regardless of UTR or not) against the corresponding gene. The open source package for microarray analysis, Limma (Smyth 2004) was used to process the data in the following steps: background correction with the minimum method, within-array normalization, between-array normalization using the Aquantile method, application of linear models for analyzing dye-swap and biological repeats, and finally the assessment of differential expression employing empirical Bayesian for multiple comparisons and the TREAT method (t-test relative to a threshold, (McCarthy and Smyth 2009) for selecting probes with biological meaningful difference).
|
| |
|
| Submission date |
Oct 08, 2010 |
| Last update date |
Feb 19, 2015 |
| Contact name |
Masanari Takamiya |
| E-mail(s) |
masanari.takamiya@kit.edu
|
| Organization name |
Karlsruhe Institute of Technology
|
| Department |
Institute of Toxicology and Genetics
|
| Lab |
Uwe Strähle
|
| Street address |
Hermann-von-Helmholtz-Platz 1
|
| City |
Kalrsruhe |
| ZIP/Postal code |
D-76344 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6457 |
| Series (2) |
| GSE24595 |
Adult zebrafish: cornea vs epidermis |
| GSE24599 |
Comparison of cornea to the surface tissues, dermis and epidermis, in adult zebrafish |
|
