gender: male and female tissue: telencephalon light: Dark cycle: NA testosterone at sacrifice: NA treatment protocol: Whole telencephalon pool of 30 birds (15 males and 15 females) supplied equally from each of 5 aviaries around the country
Treatment protocol
Birds were castrated and synchronized to a Short Day (SD) state to ensure regressed song nuclei, basal levels of Testosterone (T) typical of the non-breeding season, low song rates and unstereotyped song structure, and a low intrinsic spontaneous firing rate in the robust nucleus of the arcopalluim (RA). Then, on Day 0 of the experiment we sacrificed 6 birds. This group of birds, referred to as SD long-term, represents the steady-state of the regressed song control system and served as a baseline for all other groups. We implanted the rest of the birds with a subcutaneous Silastic capsule of T, and shifted them to Long Day (LD) photoperiod. To measure gene expression while the song control system was actively changing from a nonbreeding to a breeding phenotype, 6 birds were sacrificed on each of days 3, 7, and 21 (3LD+T, 7LD+T, and 21LD+T, respectively). By day 21, the song control system has reached its full breeding phenotype, and this time point represents the breeding-state song control system. On Day 21, the subcutaneous T capsules were removed from the remaining birds and they were shifted overnight to SD photoperiod to induce regression of the song control system back to the non-breeding state. On each of days 22 and 23 we sacrificed 6 birds (1SD-T and 2SD-T, respectively) to measure gene expression while the song control system is actively regressing. To control for circadian patterns of gene expression, all birds were sacrificed during the three hour period following their subjective dawn (i.e., lights on).
Extracted molecule
total RNA
Extraction protocol
Experimental samples: Total RNA was extracted using Absolutely RNA Microprep kit (Stratagene). 200ng total RNA was double-amplified using MessageAmp II aRNA 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.
gender: male tissue: HVC light: Dark cycle: 8:16 testosterone at sacrifice: No treatment protocol: After castration and photoperiod synchronization, birds were implanted with subcutaneous Testosterone (T), shifted to Long Days (LD) for 21 days, then T was removed and the birds were shifted back to Short Days (SD), and euthanized after 2 days.
Treatment protocol
Birds were castrated and synchronized to a Short Day (SD) state to ensure regressed song nuclei, basal levels of Testosterone (T) typical of the non-breeding season, low song rates and unstereotyped song structure, and a low intrinsic spontaneous firing rate in the robust nucleus of the arcopalluim (RA). Then, on Day 0 of the experiment we sacrificed 6 birds. This group of birds, referred to as SD long-term, represents the steady-state of the regressed song control system and served as a baseline for all other groups. We implanted the rest of the birds with a subcutaneous Silastic capsule of T, and shifted them to Long Day (LD) photoperiod. To measure gene expression while the song control system was actively changing from a nonbreeding to a breeding phenotype, 6 birds were sacrificed on each of days 3, 7, and 21 (3LD+T, 7LD+T, and 21LD+T, respectively). By day 21, the song control system has reached its full breeding phenotype, and this time point represents the breeding-state song control system. On Day 21, the subcutaneous T capsules were removed from the remaining birds and they were shifted overnight to SD photoperiod to induce regression of the song control system back to the non-breeding state. On each of days 22 and 23 we sacrificed 6 birds (1SD-T and 2SD-T, respectively) to measure gene expression while the song control system is actively regressing. To control for circadian patterns of gene expression, all birds were sacrificed during the three hour period following their subjective dawn (i.e., lights on).
Extracted molecule
total RNA
Extraction protocol
Experimental samples: Total RNA was extracted using Absolutely RNA Microprep kit (Stratagene). 200ng total RNA was double-amplified using MessageAmp II aRNA 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
The R/Bioconductor package limma was used with print-tip loess for within array normalization and the Aquantile method for the between array normalization. Data were not background-adjusted.
