One-day-old white Leghorn chicks (Moyer's Chick, Inc., Quakertown, PA) were maintained under a 12-hour light–dark cycle, under incandescent lighting (General Electric, Fairfield, CT) with irradiance of approximately 1600 μW/cm2
at chick eye level. They received food (Purina Chick Chow, Indianapolis, IN) and water ad libitum. When the chicks were 1 week of age and under inhalation ether anesthesia, a ring-shaped piece of Velcro was secured to the periorbital feathers of one eye with cyanoacrylate glue; experimental eyes were alternated between right and left in each series of chicks. Within 1 hour of the onset of the light phase on the next day and without sedation, either a +15- or –15-D clear 12-mm diameter PMMA (polymethyl methacrylate) contact lens (ABB CONCISE Optical Group LLC, Marshfield, MA) was secured to the experimental eye with complementary ring-shaped Velcro; the contralateral eye was not fitted with a lens and served as the within-subject control. After 6 hours or 3 days of spectacle lens wear (n
= 8 for each time and for either [+] or [−] lens wear), the chicks were killed by decapitation. For 6 hours of lens wear, the chicks were killed at 6 to 7 hours into the light phase, the lenses having been applied at the onset of the light phase. For 3 days of lens wear, the chicks were killed at 2 to 3 hours into the light phase. The ocular effects of spectacle lens wear by chicks are well characterized,9
and ocular refractions and eye measurements were not obtained to avoid potential anesthesia effects and to minimize postmortem mRNA degradation. As quickly as possible, the eyes were enucleated and opened at the equator; the retina/RPE was dissected together from the lens-wearing and contralateral control eyes. The tissues were individually frozen and stored in liquid nitrogen until processed. The microarray targets (as cDNA) were prepared from total RNA from each eye separately without pooling, using six chicks from each of the four experimental groups.
To verify the refractive responses, two additional groups of day-old white Leghorn chicks (Charles River Laboratories, Preston, CT; n
= 6/cohort) were reared in identical conditions. After 3 days of unilateral +15- or −15-D lens wear, they were anesthetized with a mixture of ketamine (20 mg/kg) and xylazine (5 mg/kg), and both eyes were measured by refractometry and ultrasound, as described elsewhere.22
While still under anesthesia, the chicks were killed by decapitation. The experiments conformed both to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and to the University of Pennsylvania Institutional Animal Care and Use Committee approval.
RNA was isolated from each preparation (Trizol reagent; Invitrogen, Carlsbad, CA) followed by purification and DNase treatment on RNeasy columns (Qiagen, Inc., Valencia, CA). To quantify the RNA and determine its purity, we measured the samples on a spectrophotometer (ND-1000 UV-Vis; NanoDrop Technologies, Wilmington, DE), with 260/280 nm absorbance ratios between 1.8 and 2.1. To evaluate RNA integrity further, an aliquot of each RNA sample was loaded onto an RNA chip (6000 Nano Laboratory-Chip) and placed in a bioanalyzer (model 2100; Agilent Technologies, Santa Clara, CA). RNA integrity was verified by electropherograms and gel image analysis to visualize the intact ribosomal bands using the system software. Aliquots of the RNA samples were stored individually at −80°C.
Microarray Target Preparation and Hybridization
Microarray services were provided by the Penn Microarray Facility of the University of Pennsylvania School of Medicine. All protocols were conducted as described in the manufacturers' manuals (Ovation Manual, NuGen Technologies, Inc., San Carlos, CA; GeneChip Expression Analysis Technical Manual, Affymetrix Inc., Santa Clara CA). Briefly, 100 ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by a poly(T) oligomer that incorporated a synthetic RNA sequence. Second-strand cDNA synthesis was followed by ribo-SPIA (Single Primer Isothermal Amplification, NuGEN Ovation kit) for linear amplification of each transcript, and the resulting cDNA was fragmented, assessed with the bioanalyzer, and biotinylated. cDNA yields ranged from 7.1 to 10.2 μg, and 3.75 μg was added to hybridization cocktails (Affymetrix), heated at 99°C for 2 minutes, and hybridized for 16 hours at 45°C to chicken gene microarrays (Chicken Genome GeneChips; Affymetrix) (http://www.osa.sunysb.edu/udmf/ArraySheets/chicken_datasheet.pdf
). The microarrays were then washed at low (6× SSPE) and high (100 mM MES and 0.1 M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.
Hybridization signals were quantified (Command Console and Expression Console; Affymetrix) for each probe; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. Probe intensities were exported in .cel file format (Affymetrix).
The .cel files were imported into genomics software (Genomics Suite, ver. 6.4; Partek Inc., St. Louis, MO). To permit comparison between arrays, RMA (robust multiarray average) was applied to yield background-adjusted, normalized, log2-transformed signal intensities. We performed a three-way, mixed-model analysis of variance (ANOVA; factors: time, [6 hours, 3 days]; lens, −, 0, + [i.e., minus, no or plus lens]; chick ID, [random effect], with an interaction term [time × lens]). With the ANOVA, four pairwise comparisons were also calculated: 6 hours, + vs. 0; 6 hours, − vs. 0; 3 days, + vs. 0; and 3 days, − vs. 0. All resulting P values were corrected for multiple comparisons using the Benjamini-Hochberg step-up method to yield false discovery rates (FDRs), as implemented in the genomics software (Partek). An FDR of <10% was considered significant and was used as the primary cutoff criterion for identifying differentially expressed transcripts. For the pairwise comparisons, fold-change in gene expression was also calculated, comparing the lens-wearing to contralateral control eye.
Using Affymetrix probeset identifiers, two web-based tools were used to evaluate the differentially expressed transcripts meeting the above <10% FDR statistical criterion. VENNY (http://bioinfogpcnbcsices/tools/venny/indexhtml
) was used to generate the Venn diagram of the overlapping transcripts between the two lens conditions and two times (). To evaluate potential networks and pathways implicated by the differentially expressed transcripts, we used pathway analysis (IPA ver. 8.6-3003, build 92475; Ingenuity Systems, Redwood, CA; http://www.ingenuity.com/
) with the following general analysis settings: chicken genome array as the reference set, direct and indirect relationships, endogenous chemicals included, 35 molecules/network, 25 networks, all data sources and species, nervous system tissues/CNS (central nervous system) cell lines with a relaxed filter on the molecules, and their relationships. For conformity in data reporting, we used the Affymetrix notations for gene symbols throughout.
Figure 1. Distinct and overlapping differentially expressed transcripts. Venn diagram for the distinct and overlapping differentially expressed retina/RPE transcripts for chicks wearing a unilateral +15- or –15-D spectacle lens for 6 hours or 3 days. The (more ...)
Real-Time Quantitative RT-PCR
To ascertain the reliability of the microarray results, we conducted both biological and technical validations of the expression profiling for selected known genes, using real-time quantitative reverse transcription-polymerase chain reaction (qPCR). Biological replicates are assays on animals different from those subjected to microarray analysis but reared and processed contemporaneously; here, we used the retina/RPE from the two other chicks reared contemporaneously under each of the four conditions. Technical validations are measurements of aliquots from the same biological specimen with a different measuring technique; here, for technical validations, we used residual RNA from the retina/RPE assayed by microarray. For both types of qPCR validations, 1 μg of retina/RPE RNA was converted into cDNA (Superscript III First Strand cDNA Synthesis Supermix for qPCR; Invitrogen).
For the biological validations, we selected transcripts of known genes with a fold-change of approximately ≥1.4 in either the up- or downregulated direction, including some genes that were differentially expressed under more than one condition. Within that group, we included vasoactive intestinal peptide (VIP), noggin (NOG), and bone morphogenetic protein 2 (BMP2), because of their relation to findings in our prior profiling of the retina/RPE in form-deprivation myopia.16
For technical validations, four genes/conditions with comparatively high microarray fold-changes were selected from among the genes validated in the biologically independent samples; and qPCR assays were conducted with cDNA from both retinas of all six chicks studied by microarray with the same primers (see Results for genes/conditions). For the technical validations, the normalized expression value for each gene in the experimental retina/RPE was compared to that of its contralateral control using a paired t
For qPCR, primer sets optimized for chicken sequences were purchased (Quantitect Primer Assays; Qiagen) for BMP2 (NM_204358); dual-specificity phosphatase 4 (DUSP4; NM_204838); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; NM_204305); myosin, heavy chain 13, skeletal muscle (MYH13; XM_001231455); NOG (NM_204123); oxysterol binding protein 6 (OSBPL; XM_421982; XM_001233035); phosphodiesterase 3A, cGMP-inhibited (PDE3A;XM_416416); urotensin 2 domain containing (UTS2D; NM_206989; VIP (NM_205366). The primer sets are designed to amplify across intron–exon boundaries; details of size and position of amplicons on each gene of interest are available online from the manufacturer (Qiagen; www.qiagen.com/GeneGlobe
Using a real-time PCR system (model 7300; Applied Biosystems, Inc., [ABI], Foster City, CA) and 96-well plates, triplicate 30 μL PCR reactions were performed for each gene of interest using cDNA (8 ng/reaction), the gene-specific primers and master mix (QuantiFast SYBR Green RT-PCR; Qiagen). The PCR reaction comprised 40 cycles at 95°C for 15 seconds and 60°C for 32 seconds. The analyses were performed using the system software (model 7300; ABI) and followed directions provided by the manufacturer (Guide to Performing Relative Quantification of Gene Expression RT-qPCR) using the comparative ΔΔ
relative quantification method.23
Before quantitative analysis, the efficiencies of the reference gene GAPDH and the genes of interest were determined to be the same; GAPDH expression was verified as unaltered across experimental conditions.