Feed access (fed or fasted) was administered according to the individual sample descriptions. At the time of sampling, chicks were killed and the hypothalami were immediately removed and snap frozen in liquid nitrogen. Samples were stored at -80C until RNA extraction.
Growth protocol
Fertile eggs (Ross x Ross) were obtained from Allen’s hatchery (Seaford, DE) and incubated at the University of Maryland under standard conditions. The chicks for this experiment were collected within a four-hour window of hatching, and males were randomly assigned to treatment groups. All chicks were given free access to water. Chicks in the ‘Hatch’ group did not receive feed before samples were collected. Fed chicks received standard chick starter ration ad libitum, and all chicks were brooded at age appropriate temperatures in battery brooders.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from hypothalami using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The isolated RNA was quantified using the Ribogreen RNA Quantitation Kit (Molecular Probes, Eugene, OR) and RNA quality was determined using a Bioanalyzer 2100 (Agilent, Wilmington, DE). RNA was then amplified using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion, Austin, TX). One round of RNA amplification was performed using 4µg of total RNA. In vitro transcription (IVT) was performed for 14h at 37C. aRNA was quantified and analyzed for quality using the same methods as were used for total RNA.
Label
Alexa 555
Label protocol
Dye labeling was accomplished according to the instructions for dye labeling and labeled aRNA cleanup with the Amino Allyl MessageAmpTM II aRNA Amplification Kit. Twenty micrograms of aRNA were dried to a minimal volume (not overdryed) in a SpeedVac concentrator. The aRNA was then reconstituted in 9µL of coupling buffer and mixed with 11µL of prepared dye (Alexa fluor 555 carboxylic acid, succinimidyl ester and Alexa fluor 647 carboxylic acid, succinimidyl ester (Invitrogen, Eugene, OR)). Aliquots of the dye (60 µg) were dissolved in DMSO just prior to use. aRNA with dye was incubated in the dark for one h at room temperature for coupling. The incorporation of dye and concentration of aRNA were quantified with the NanoDrop spectrophotometer (Wilmington, DE) and incorporation efficiency was determined using the calculator available at www.ambion.com/tools/dye.
Pool of 160 hypothalami Pool of all samples in experiment
Treatment protocol
Feed access (fed or fasted) was administered according to the individual sample descriptions. At the time of sampling, chicks were killed and the hypothalami were immediately removed and snap frozen in liquid nitrogen. Samples were stored at -80C until RNA extraction.
Growth protocol
Fertile eggs (Ross x Ross) were obtained from Allen’s hatchery (Seaford, DE) and incubated at the University of Maryland under standard conditions. The chicks for this experiment were collected within a four-hour window of hatching, and males were randomly assigned to treatment groups. All chicks were given free access to water. Chicks in the ‘Hatch’ group did not receive feed before samples were collected. Fed chicks received standard chick starter ration ad libitum, and all chicks were brooded at age appropriate temperatures in battery brooders.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from hypothalami using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The isolated RNA was quantified using the Ribogreen RNA Quantitation Kit (Molecular Probes, Eugene, OR) and RNA quality was determined using a Bioanalyzer 2100 (Agilent, Wilmington, DE). RNA was then amplified using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion, Austin, TX). One round of RNA amplification was performed using 4µg of total RNA. In vitro transcription (IVT) was performed for 14h at 37C. aRNA was quantified and analyzed for quality using the same methods as were used for total RNA.
Label
Alexa 647
Label protocol
Dye labeling was accomplished according to the instructions for dye labeling and labeled aRNA cleanup with the Amino Allyl MessageAmpTM II aRNA Amplification Kit. Twenty micrograms of aRNA were dried to a minimal volume (not overdryed) in a SpeedVac concentrator. The aRNA was then reconstituted in 9µL of coupling buffer and mixed with 11µL of prepared dye (Alexa fluor 555 carboxylic acid, succinimidyl ester and Alexa fluor 647 carboxylic acid, succinimidyl ester (Invitrogen, Eugene, OR)). Aliquots of the dye (60 µg) were dissolved in DMSO just prior to use. aRNA with dye was incubated in the dark for one h at room temperature for coupling. The incorporation of dye and concentration of aRNA were quantified with the NanoDrop spectrophotometer (Wilmington, DE) and incorporation efficiency was determined using the calculator available at www.ambion.com/tools/dye.
Hybridization protocol
The arrays were baked at 90C for 90 minutes prior to use. Pre-treatment of slides was accomplished by incubation of slides in pre-hybridization solution (5xSSC, 0.1%SDS, and 1% BSA) for 45 min at 42C. Then slides were dipped in 2x SSC and 0.2x SSC for 5 min each. Slides were dried by centrifugation (5 min at 1000 x g). An aliquot of 5 µg of labeled, purified aRNA was fragmented in 1x fragmentation buffer (Ambion, Austin, TX) for 15 min at 70C. Stop solution was added to terminate the reaction, and samples were dried again in the SpeedVac. Pre-warmed DIG Easy Hyb solution (Roche Diagnostics Corporation, Indianapolis, IN) was used to reconstitute the samples in a 30 µL volume. Reference aRNA samples used within the same day were pooled and aliquoted. One reference pool (30 µL) and one sample (target) (30 µL) were mixed and 2.5 µL of 10mg/mL yeast tRNA and 2.5 µL of 10mg/mL salmon testes DNA (Sigma, St. Louis, MO) were added. The target/reference mixture (65 µL) was heated to 94C for 2 min for denaturation, cooled at room temperature, and loaded to the middle of a pre-treated slide held in a hybridization chamber (Corning #2551, Lowell, MA), which was overlaid with a 22 x 65 mm LifterSlip (Erie Scientific, Portsmouth, NH). Slides were then incubated for 14-16h at 42C inside a light-tight sealed box in a water bath. Following incubation, the cover slip was removed by flushing with wash solution #1 (1x SSC, 0.2% SDS, 0.5% DTT). Sequential washes were as follows: Ten min at 42C in pre-warmed wash solution #1, five min at room temp in wash solution #2 (0.1x SSC, 0.2% SDS, 0.5% DTT), and one min at room temperature in wash solution #3 (0.1x SSC, 0.5% DTT). Slides were then washed with ddH2O and dried by centrifugation. Slides were stored in 50 mL conical tubes covered in foil in the presence of nitrogen gas until scanning.
Scan protocol
GenePix 4000B microarray scanner was used to scan each slide. The image was acquired at 10 µm resolution during simultaneous scanning of two wavelengths, 532nm (green) and 635nm (red). The PMT count ratio was adjusted to 1 during preview at low resolution scanning, and a high resolution TIFF image file was then obtained.
Description
Biological Replicate 3 of 4.
Data processing
Each TIFF image was further analyzed with GenePix Pro software, generating a GenePix Report (gpr) file. GenePix report files were converted to .mev files using Express Converter software freely available from The Institute for Genomic Research (TIGR) website. Converted files were Lowess normalized using Microarray Data Analysis System (MIDAS) software, also freely available on the TIGR website.
