Free-ranging male song sparrow (Melospiza melodia morphna) were tested in the autumn (mid November, 2005 to late January, 2006; the non-breeding season) in one the following 7 sites in western Washington State (48ºN): Crescent Lake, Cherry Valley, Prison Farm, Spencer Island (all in Snohomish county), Skagit Valley (Skagit county), Pack Forest (Pierce county), Stillwater (King county). To simulate a territorial intrusion, a free-ranging territorial male (focal bird) was presented with a live caged male song sparrow decoy and recorded male conspecific songs. During the first 10 minutes of the STI, the following behaviors were recorded from the focal bird: 1) number of songs, 2) number of flights directed at the decoy, 3) closest approach to the decoy, and 4) time spent within 5 meters (m) of the decoy. The STI was continued for additional 20 min and then the bird was quickly captured with a mist net. Immediately after capture, the bird was anesthetized with isoflurane (10%) and decapitated. Hypothalamus was quickly dissected out and quickly frozen on dry ice and subsequently stored at -80 ºC. Due to the location and structural association of the pars tuberalis in avian species, it was impossible to accurately dissect out pars tuberalis from the hypothalamus, therefore all samples were collected along with pars tuberalis for consistency; however, other parts of the pituitary were removed.
Extracted molecule
total RNA
Extraction protocol
Experimental samples: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; 0.5µg 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 46C for 16 h in the presence of 400 U SuperScript III 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.
season: NA intrustion treatment: NA brain region: telencephalon gender: male and female
Treatment protocol
Whole telencephalon pool of 30 birds (15 males and 15 females) supplied equally from each of 5 aviaries around the country
Extracted molecule
total RNA
Extraction protocol
Experimental samples: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; 0.5µg 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 46C for 16 h in the presence of 400 U SuperScript III 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 Chamber, and incubated O/N at 42ûC. 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.
Description
print batch: 5/31/2006
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.
