Sample preparation and RNA extraction
Experimental animals for this study were maintained at the University Animal Farm, Seoul National University, Korea. All experimental procedures were performed at the affiliated laboratories of the university.
Eggs were brought to the lab within 1 to 3 h of oviposition for stage X embryos [4]. Developing embryos under relative humidity of 60–70% at 37.8°C were staged according to the Hamburger and Hamilton (HH) classification system [6]. Thus, Eyal-Giladi and Kochav (EK) stages X, Hamburg and Hamilton (HH) stage 3, HH stage 6, and HH stage 9 correspond to embryos at 0 and 12 h, 24 h, and 30 h of incubation, respectively.
Total RNA from embryos at each stage was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. RNA quality was checked by agarose gel electrophoresis and RNA quantity was determined by spectrophotometry at 260 nm. RNA was then further purified using the RNeasy kit (Qiagen, Valencia, CA). The total RNA was used for gene expression analysis on an Affymetrix GeneChip Chicken Genome Array (Affymetrix, Santa Clara, CA), containing 38,535 probes.
Generation of microarray data
All experiments were performed in triplicate. The generation of GeneChip data from the embryos of stage X and HH stages 3 was performed by Seoulin Bioscience Corporation (Seoul, Korea). Specifically, total RNA (about 5 μg) from stage X and HH stages 3 embryos was used for labelling. Probe synthesis from total RNA samples, hybridization, detection, and scanning were performed according to standard protocols from Affymetrix. Briefly, cDNA was synthesized using the One-Cycle cDNA Synthesis Kit (Affymetrix). Single-stranded (ss) cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo (dT) primers at 42°C for 1 h. Double-stranded (ds) cDNA was obtained using DNA ligase, DNA polymerase I, and RNase H at 16°C for 2 h, followed by T4DNA polymerase at 16°C for 5 min. After cleanup using a Sample Cleanup Module (Affymetrix, Santa Clara, CA), ds cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the GeneChip IVT Labeling Kit (Affymetrix) in the presence of biotin-labeled CTP and UTP. Then the biotin-labeled IVT-RNA was fragmented and hybridized to the chicken genome GeneChip array at 45°C for 16 h, according to the manufacturer's instructions. After hybridization, the arrays were washed in a GeneChip Fluidics Station 450 with a non-stringent wash buffer at 25°C, followed by a stringent wash buffer at 50°C. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner, controlled by GeneChip Operating Software (GCOS; Affymetrix).
Microarray data analysis
The quality of the array image was assessed as described in the GeneChip expression analysis manual (Affymetrix). All arrays were processed to determine the "robust multi-array average" (RMA; [22]) using the "affy" software package [23]. Expression values were computed in detail from raw CEL files by applying the RMA model of probe-specific correction for perfect-match probes. These corrected probe values were then subjected to quantile normalization, and a median polish was applied to compute one expression measure from all probe values. Resulting RMA expression values were log2-transformed. Clustering from the normalized expression data of all probes was performed and displayed using Cluster and TreeView software [24].
Individual gene expression levels from embryos at stage X and HH stages 3 were compared using an unpaired Student's t-test. The Benjamini-Hochberg correction for false discovery rate (FDR) was used for all probe-level normalized data. We selected differentially expressed genes that met the following criteria: a change in expression of at least twofold when comparing the means of the two groups and a FDR-adjusted P value of less than 0.01 according to the unpaired Student's t-test. Gene Ontology annotation was conducted using NetAffx tool provided by the Affymetrix [25]. Further analysis for functional annotation clustering was performed using the DAVID database [26] [see Additional file 1].
Quantitative RT-PCR
To confirm the GeneChip expression data and the relative gene expression profiling, quantitative RT-PCR was performed at the developmental stages. Based on microarray data, we selected 36 genes and primers were designed using the Primer 3 software [27] on sequences from the GenBank database (Table 3, 4). For quantitative RT-PCR, we extended embryo development stages from stage X to stage 9 and so prepared total mRNAs from 4 developmental points; stage X (0 h), HH stage 3 (12 h), HH stage 6 (24 h), and HH stage 9 (30 h). sscDNA was synthesized from total RNA (1 μg) using the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). The cDNA was serially diluted fivefold and was quantitatively equalized for PCR amplification. Quantitative RT-PCR was performed using the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) and SYBR Green I (Sigma, St. Louis, MO). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was run simultaneously as a control and used for normalization. Non-template control wells with no cDNA were included as negative controls. Each test sample was run in triplicate.
Following the standard curve method, the expression quantities of the examined genes were determined using the standard curves and the Ct values, and were normalized using GAPDH expression quantities. The PCR conditions were 94°C for 3 min, followed by 40 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, using a melting curve program (increasing temperature from 55 to 95°C with a heating rate of 0.5°C per 10 sec) and continuous fluorescence measurement. Sequence-specific products were identified by generating a melting curve. The Ct value represents the cycle number at which a fluorescent signal rises statistically above background and the relative quantification of gene expression was analyzed by the2-ΔΔCt method [28]. Based on the quantification RT-PCR results, hierarchical clustering was performed, and the relative quantification of gene expression was normalized to the Ct of stage X as a control group.