|
| Status |
Public on Jul 01, 2016 |
| Title |
Brain 18 14d 03 No21 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brain cold treatment for 14 d
|
| Organism |
Danio rerio |
| Characteristics |
The AB strain of zebrafish (D. rerio) was originally obtained from the University of Oregon, and were kept in the zebrafish stock center at Academia Sinica, Taipei, Taiwan.
|
| Biomaterial provider |
Academia Sinica
|
| Treatment protocol |
Adult zebrafish were acclimated to 18 C with a gradually reduced temperature at a gradient of 4 C/h in order to prevent temperature shock and reduce mortality.
|
| Growth protocol |
The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2006083).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues dissected from 10 individuals were pooled as a sample and then homogenized in 0.8 ml Trizol reagent (Invitrogen, Carlsbad, CA). After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and Bioanalyzer (Aglient Technologies, Foster City, CA).
|
| Label |
Alexa 647 dye
|
| Label protocol |
cDNA probes were synthesized and purified by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and MinElute PCR purification kit (Qiagen) and were labeled with Alexa 647 dye (cold treatment groups).
|
| |
|
| Channel 2 |
| Source name |
Brain normal temperature treatment for 14 d
|
| Organism |
Danio rerio |
| Characteristics |
The AB strain of zebrafish (D. rerio) was originally obtained from the University of Oregon, and were kept in the zebrafish stock center at Academia Sinica, Taipei, Taiwan.
|
| Biomaterial provider |
Academia Sinica
|
| Treatment protocol |
Adult zebrafish were incubated in normal temperature as a control group.
|
| Growth protocol |
The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2006083).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues dissected from 10 individuals were pooled as a sample and then homogenized in 0.8 ml Trizol reagent (Invitrogen, Carlsbad, CA). After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and Bioanalyzer (Aglient Technologies, Foster City, CA).
|
| Label |
Alexa 555 dye
|
| Label protocol |
cDNA probes were synthesized and purified by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and MinElute PCR purification kit (Qiagen) and were labeled with Alexa 555 dye (cold treatment groups).
|
| |
|
| |
| Hybridization protocol |
The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 °C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 µg/µl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each).
|
| Scan protocol |
Scanning was performed with a Scanarray Gx scanner (PerkinElmer, Waltham, MA). The acquired images were analyzed using ScanArray Express 3.0 (PerkinElmer, Waltham, MA)and Genespring software (Aglient Technologies, Foster City, CA).
|
| Description |
The commercial zebrafish 14K oligonucleotides set (MWG
Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufacture’s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website.
|
| Data processing |
The measurements of spots were filtered by flags, and the Lowess normalization was performed after subtraction of the median background. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples. The differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5), and then the Significant Analysis of Microarray method (SAM 3.02) was used to determine statistical significances.
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| |
|
| Submission date |
Mar 26, 2008 |
| Last update date |
Jul 01, 2016 |
| Contact name |
Ming Yi Chou |
| E-mail(s) |
eleber@gmail.com
|
| Phone |
886-227899521
|
| Organization name |
Acdemia Sinica
|
| Department |
Cellular and Organismic Biology
|
| Lab |
Fish ecophysiology
|
| Street address |
128 Sec. 2 Acdemia Rd.
|
| City |
Taipei |
| State/province |
Taiwan |
| ZIP/Postal code |
115 |
| Country |
Japan |
| |
|
| Platform ID |
GPL5182 |
| Series (1) |
| GSE10958 |
Comparisons of gene expressions in zebrafish (Danio rerio) brain and gill after cold acclimation |
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