|
| Status |
Public on May 31, 2012 |
| Title |
Adult female NCM after 90 min novel song playback replicate 2 [e10] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NCM.90min
|
| Organism |
Taeniopygia guttata |
| Characteristics |
brain region: NCM
song playback experience: 90 minutes after onset of novel song
gender: Female
age: Adult
|
| Treatment protocol |
Adult female zebra finches were acoustically isolated overnight, then 30' of playback was presented that consisted of a repeated medley of 3 zebra finch songs over 15 followed by a 45 interval of silence. 30 min samples were collected at this time; birds remained in the chamber for an additional 60 min to collect the 90 min samples. Silence control birds were acoustically isolated but heard no playback the next day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental: Total RNA was extracted from NCM and L2a tissue punches using the Absolutely RNA kit (Stratagene). 500ng were amplified one round using the MessageAmp II kit (Ambion). 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 |
universal SoNG reference
|
| Organism |
Taeniopygia guttata |
| Characteristics |
brain region: Telencephalon
song playback experience: NA
gender: Male and female
age: Adult
|
| Treatment protocol |
Adult female zebra finches were acoustically isolated overnight, then 30' of playback was presented that consisted of a repeated medley of 3 zebra finch songs over 15 followed by a 45 interval of silence. 30 min samples were collected at this time; birds remained in the chamber for an additional 60 min to collect the 90 min samples. Silence control birds were acoustically isolated but heard no playback the next day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental: Total RNA was extracted from NCM and L2a tissue punches using the Absolutely RNA kit (Stratagene). 500ng were amplified one round using the MessageAmp II kit (Ambion). 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) |
| GSE36739 |
The impact of experience-dependent and independent factors on gene expression in songbird brain [e10] |
| GSE36748 |
The impact of experience-dependent and independent factors on gene expression in songbird brain |
|
