|
| Status |
Public on May 31, 2012 |
| Title |
AL of Adult male familiar song replicate 3 [e11] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
universal SoNG reference
|
| Organism |
Taeniopygia guttata |
| Characteristics |
brain region: Telencephalon
song playback experience: NA
gender: Male and female
age: Adult
|
| Treatment protocol |
Adult male zebra finches (Taeniopygia guttata) were acoutically isolated in playback chambers overnight. The next day (day1), one group of birds heard 30 minutes of a novel song (e.g. SongA), sacrificed and tissue collected and flash frozen. Silence control birds were taken at the equivalent time of day1 as novel song birds, but heard no song playback. The birds hearing familiar song were isolated as the others, but on the first day heard 180 min of SongA, remained in the chambers a second night, and heard 30 min of SongA again the follwing day (day2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental samples: Total RNA was extracted using RNAqueous Mini kit (Ambion) and DNase treated with TURBO DNase (Ambion), and cleaned on a spin column. 500ng total RNA was amplified using Agilent's Low RNA input linear amplification kit. Universal reference: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; equal amounts of total RNA from each bird was pooled; total RNA was amplified from this pool using Agilent's Low RNA input linear amplification kit to create sufficient aRNA for 5000 assays.
|
| Label |
Cy5
|
| Label protocol |
1 µg of aRNA was primed with 3 µl of 100 µM random hexamer primers at 70C for 10 min, then reversed transcribed at 42C for 16 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM dATP, dCTP, dGTP, 60µM dTTP and 40µM aa-dUTP; RNA was hydrolyzed in the presence of NaOH and EDTA and samples were cleaned up on a spin column. Finally, Cy3 and Cy5 dye esters (GE Healthcare) were coupled to aa-dUTP residues in NaCO3 buffer for 2 hrs at room temp, then neutralized and cleaned on spin columns.
|
| |
|
| Channel 2 |
| Source name |
Familiar song
|
| Organism |
Taeniopygia guttata |
| Characteristics |
brain region: AL (auditory lobule)
song playback experience: Trained 180 min with song on day1, heard 30 min of same song on day2
gender: Male
age: Adult
|
| Treatment protocol |
Adult male zebra finches (Taeniopygia guttata) were acoutically isolated in playback chambers overnight. The next day (day1), one group of birds heard 30 minutes of a novel song (e.g. SongA), sacrificed and tissue collected and flash frozen. Silence control birds were taken at the equivalent time of day1 as novel song birds, but heard no song playback. The birds hearing familiar song were isolated as the others, but on the first day heard 180 min of SongA, remained in the chambers a second night, and heard 30 min of SongA again the follwing day (day2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental samples: Total RNA was extracted using RNAqueous Mini kit (Ambion) and DNase treated with TURBO DNase (Ambion), and cleaned on a spin column. 500ng total RNA was amplified using Agilent's Low RNA input linear amplification kit. Universal reference: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; equal amounts of total RNA from each bird was pooled; total RNA was amplified from this pool using Agilent's Low RNA input linear amplification kit to create sufficient aRNA for 5000 assays.
|
| Label |
Cy3
|
| Label protocol |
1 µg of aRNA was primed with 3 µl of 100 µM random hexamer primers at 70C for 10 min, then reversed transcribed at 42C for 16 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM dATP, dCTP, dGTP, 60µM dTTP and 40µM aa-dUTP; RNA was hydrolyzed in the presence of NaOH and EDTA and samples were cleaned up on a spin column. Finally, Cy3 and Cy5 dye esters (GE Healthcare) were coupled to aa-dUTP residues in NaCO3 buffer for 2 hrs at room temp, then neutralized and cleaned on spin columns.
|
| |
|
| |
| Hybridization protocol |
Samples were suspended in SlideHyb#1 (Ambion), applied to slides in Corning Hybridization Chambers, and incubated O/N at 42C. After hybridization, the slides were washed sequentially in 1X SSC, 0.2% SDS; 0.1X SSC, 0.2% SDS; and 0.1X SSC for 5 minutes each, and spun dry.
|
| Scan protocol |
Scanned on an Axon GenePix 4000B scanner
Images were quantified using Axon GenePix 6.0.
|
| Data processing |
Data were background-subtracted and values < 0.5 were set to 0.5. Spots with -100 flags were filtered out before print-tip loess normalization. A scale between-array normalization was performed, then arrays with the reference in the Cy5 channel were inverted by multiplying by -1 to make all log ratios test/reference.
|
| |
|
| Submission date |
Mar 23, 2012 |
| Last update date |
May 31, 2012 |
| Contact name |
Kirstin Replogle |
| E-mail(s) |
replogle@igb.uiuc.edu
|
| Organization name |
University of Illinois
|
| Street address |
1206 W Gregory Drive
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL9554 |
| Series (2) |
| GSE36740 |
The impact of experience-dependent and independent factors on gene expression in songbird brain [e11] |
| GSE36748 |
The impact of experience-dependent and independent factors on gene expression in songbird brain |
|
| Relations |
| Reanalysis of |
GSM885655 |
