White Leghorn Gender not determined 10 days old Young chicks are extremely difficult to sex from external signs. Post-mortem dissections were not performed. The chicks were purchased unsexed.
Biomaterial provider
CBT Farms, Chestertown, MD
Treatment protocol
One-day old white Leghorn chicks were housed in brooders with a 12 hr light:dark cycle, using General Electric chroma 50 fluorescent lighting with irradiance of approximately 50μW/cm2 at chick eye level. They received Purina Chick Chow food and water ad libitum. At one week of age and at the onset of the light phase, the chicks were anesthetized with inhalation ether, and a unilateral translucent white plastic goggle was glued to the periorbital feathers to induce ipsilateral form-deprivation myopia, alternating between the left or right eye. After either 6 hrs (n=8) or 3 days (n=8) of goggle wear, the chicks were killed by decapitation. The enucleated eyes were opened at the equator, and the retina/RPE was dissected together from both the goggled and control eyes. The tissues were individually frozen and stored in liquid nitrogen until processed.
Extracted molecule
total RNA
Extraction protocol
Each frozen retina/RPE sample was ground under liquid nitrogen, using a mortar and pestle. RNA was then isolated from each preparation using Trizol (Invitrogen, Carlsbad, CA) followed by purification and DNase treatment on RNeasy columns (Qiagen,Inc, Valencia, CA). The samples were measured on a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE) for quantification and purity, with 260/280nm absorbance ratios between 1.8 and 2.1. To evaluate RNA integrity further, an aliquot of each RNA sample was loaded onto an RNA 6000 NanoLab Chip and placed in an Agilent 2100 Bioanalyzer (Agilent Technologies, SantaClara, CA); suitable RNA integrity was established from the electropherograms and gel-images generated by the Bioanalyzer software (data not shown). Aliquots of the RNA samples were stored individually at –80º C.
Label
Biotin
Label protocol
see manufacturer's website
Hybridization protocol
see manufacturer's website
Scan protocol
see manufacturer's website Scanner: GeneChip 3000 7G
Description
Microarray targets were prepared using total RNA from each eye of six chicks from each of the two experimental groups. All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Inc., Santa Clara, CA). Briefly, 3μg of total RNA from each eye was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 hrs at 45ºC to one of twenty-four Affymetrix GeneChip Chicken Genome Arras. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. Using a GeneChip 3000 7G scanner, the fluorescence signal (570nm excitation) was recorded at 2.5μm resolution. Data were collected using GCOS software, (v1.4, Affymetrix), and probe level intensities were exported as .cel files for subsequent summarization and analysis.
Data processing
Affymetrix .cel (probe intensity) files were exported from GCOS, and processed using Stratagene’s ArrayAssist Lite v3.1 (Stratagene, La Jolla, CA ) to yield .chp files containing Robust Multichip Analysis (RMA) expression values and Affymetrix flags (Present, Absent, Marginal) for each array. The normalized microarray signal intensities were then analyzed by the Significance Analysis of Microarrays approach.(SAM). For the retinas of chicks with 6 hours of unilateral form-deprivation, the SAM analysis indicated few differentially expressed genes; and a 50% false discovery rate was arbitrarily set to yield a list of 112 candidate genes. For the retinas of chicks with 3 days of unilateral form-deprivation, the SAM false discovery rate was set arbitrarily at 14% to yield 296 (i.e., approximately 300) candidate genes.
