|
| Status |
Public on Jan 16, 2010 |
| Title |
Area X, silent 1hr, Replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Area X, silent 1hr
|
| Organism |
Taeniopygia guttata |
| Characteristics |
tissue: Brain
gender: Male
age: >120 days
|
| Treatment protocol |
In the morning before singing or immediately after singing for 0.5, 1, 2, 3, 4, 5, 6, or 7hrs, brains were quickly dissected. For the singing groups, we used birds that sang continuously >25 bouts per 0.5 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Picopure RNA Extraction kit (Molecular Devices, Sunnyvale, CA) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer (Nanodrop Wilmington, DE) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). From 1.5-2ng of total RNA, amplified cDNA was prepared using the WT-Ovation Pico System (Nugen, San Carlos, CA) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
2.2 ugs of purified cDNA was labeled and fragmented using FL-Ovation Cy3 labeling and fragmentation kit according to the manufacturers instructions (Nugen). 2.2 ugs of purified cDNA was labeled and fragmented using FL-Ovation Cy3 labeling and fragmentation kit according to the manufacturers instructions (Nugen). Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Labeled cDNA was dried in a speed-vac and resuspended in 25ul of hybridization buffer that contained 16% formamide, and 1X Agilent Blocking buffer. Samples were then denatured at 95 degrees for 3 mins and 18uLs of each samples was loaded to each array. Each micrroarray slide contained 4 arrays (4X44K) that were hybridized using a special A4 clamp to hold Agilent 4 x 44K arrays in Maui A4 mixers. The mixer was held at 55oC for at least 20 hrs, with the humidity chamber positioned over arrays.The following day, slides were rinsed in pre-heated (42oC). They were then shaken in Agilent Wash 1 buffer solution at RT X 1min and then Agilent Wash 2 at 37C for 1min. The slides were then slowly removed from the Wash buffer to minimize droplet formation.
|
| Scan protocol |
Slides were scanned immediately after washing on the GenePix 4000 Microarray Scanner and Agilent's Feature Extraction Software v9.5.1 using one color scan setting for 4x44k array slides (Scan resolution was 5um, Dye channel was set to 532 [Green] and PMT to 520).
|
| Description |
Silent control sample for gene expression after singing
54 male zebra finch birds were isolated overnight in sound attenuation chambers on a 12/12 light:dark cycle with ad lib access to food and water.
|
| Data processing |
The data was normalized with variance stabilization for one-color data based on Huber et al 2002 [PMID 12169536]. Microarrays with a low percentage of probes above background and high standard deviation from the median were removed.
|
| |
|
| Submission date |
Jan 14, 2010 |
| Last update date |
Jan 15, 2010 |
| Contact name |
Osceola Whitney |
| E-mail(s) |
whitney@neuro.duke.edu
|
| Phone |
919-681-1681
|
| Fax |
919-681-0877
|
| URL |
http://songbirdtranscriptome.net/
|
| Organization name |
Duke University Medical Center
|
| Department |
Neurobiology
|
| Lab |
Erich Jarvis Lab
|
| Street address |
Research Drive, Bryan Research Building, Room 401E
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL9856 |
| Series (1) |
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