|
| Status |
Public on Jun 09, 2010 |
| Title |
wt7_red vs. XOPS-mCFP7_green |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
retina from XOPS-mCFP adult zebrafish
|
| Organism |
Danio rerio |
| Characteristics |
strain: XOPS-mCFP
age: 10-11 months old
tissue: retina
|
| Growth protocol |
Rearing, breeding, and staging of zebrafish were performed in accordance with established methods for zebrafish animal husbandry. Wild-type zebrafish were inbred descendants of the Ekwill strain, originally obtained from the Ekkwill fish farm (Gibsonton, FL). The Tg(XRho:gap43-mCFP)q13 transgenic line, referred to here as XOPS-mCFP, has been described previously. It harbors a fluorescent mCFP reporter transgene under the control of the Xenopus rhodopsin promoter, the expression of which is toxic to the rod photoreceptors.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the microarray analysis, four wild type and four XOPS-mCFP adults (10-11 months of age) were sacrificed by immersion in MS-222 (Tricaine, Sigma). The right eye was dissected and the sclera, choroid and lens were removed. The retinas were transferred to microcentrifuge tubes containing RNA later (Ambion/Applied Biosystems, Austin, TX). Individual retinas were not pooled. Total RNA was prepared from each sample using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
RNA samples were submitted to the Gene Expression Core Facility at the University of Florida’s Interdisciplinary Center for Biotechnology Resarch (ICBR). RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Preparation of cDNA, generation and fluorescent labeling of cRNA, and chip hybridization were performed by the ICBR according to standard protocols.
|
| |
|
| Channel 2 |
| Source name |
retina from wild-type adult zebrafish
|
| Organism |
Danio rerio |
| Characteristics |
strain: wild type
age: 10-11 months
tissue: retina
|
| Growth protocol |
Rearing, breeding, and staging of zebrafish were performed in accordance with established methods for zebrafish animal husbandry. Wild-type zebrafish were inbred descendants of the Ekwill strain, originally obtained from the Ekkwill fish farm (Gibsonton, FL). The Tg(XRho:gap43-mCFP)q13 transgenic line, referred to here as XOPS-mCFP, has been described previously. It harbors a fluorescent mCFP reporter transgene under the control of the Xenopus rhodopsin promoter, the expression of which is toxic to the rod photoreceptors.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the microarray analysis, four wild type and four XOPS-mCFP adults (10-11 months of age) were sacrificed by immersion in MS-222 (Tricaine, Sigma). The right eye was dissected and the sclera, choroid and lens were removed. The retinas were transferred to microcentrifuge tubes containing RNA later (Ambion/Applied Biosystems, Austin, TX). Individual retinas were not pooled. Total RNA was prepared from each sample using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
RNA samples were submitted to the Gene Expression Core Facility at the University of Florida’s Interdisciplinary Center for Biotechnology Resarch (ICBR). RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Preparation of cDNA, generation and fluorescent labeling of cRNA, and chip hybridization were performed by the ICBR according to standard protocols.
|
| |
|
| |
| Hybridization protocol |
Fluorescently labeled wild-type and XOPS-mCFP cRNA were co-hybridized to an Agilent 4 x 44k zebrafish microarray containing 42,990 probes representing roughly 21,500 zebrafish ESTs. Eight hybridizations (representing four individual wild-type and XOPS-mCFP retinas) were performed; hybridizations included a reciprocal dye-swap control to account for biological, sample preparation, and technical variability.
|
| Scan protocol |
Microarray scanning and image processing were performed by the ICBR using Agilent's Feature Extraction software. The intensity of each spot was summarized by the mean pixel intensity. After background subtraction, raw data were normalized by Lowess transformation.
|
| Data processing |
After background subtraction, raw data were normalized by Lowess transformation. A 2-way ANOVA comparison was employed for differential expression analysis using the Analyse-It tool (Microsoft Excel). The p-values were adjusted using the Benjamini and Hochberg method to control for false discovery rate. Differentially expressed genes were ranked by the adjusted p-values; genes with a p-value of less than 0.005 were considered as differentially expressed between wild-type and XOPS-mCFP retinas.
|
| |
|
| Submission date |
Jun 08, 2010 |
| Last update date |
Jun 08, 2010 |
| Contact name |
Ann Caroline Morris |
| E-mail(s) |
ann.morris@uky.edu
|
| Phone |
859-257-8823
|
| Organization name |
University of Kentucky
|
| Department |
Department of Biology
|
| Street address |
215 T.H. Morgan Building
|
| City |
Lexington |
| State/province |
KY |
| ZIP/Postal code |
40506-0225 |
| Country |
USA |
| |
|
| Platform ID |
GPL6563 |
| Series (1) |
| GSE22221 |
Comparison of retinal gene expression in wild-type and XOPS-mCFP (rod degeneration) transgenic zebrafish |
|
