white Leghorn Gender: female Age: stage 29 (6-6.5 day incubation) Tissue: brain
Extracted molecule
total RNA
Extraction protocol
samples of brains at stage 29 were homogenized and total RNA from both genetic male and female brains was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality was checked on an agarose gel and RNA quantity was determined by spectrophotometry at 260/280-m absorption ratios and then purified with an RNeasy kit (QIAGEN, Valencia, CA, USA).
Label
biotin
Label protocol
cDNA was synthesized using the One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA). Single-stranded 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 T4 DNA polymerase at 16°C for 5 min. After cleanup with a Sample Cleanup Module (Affymetrix), ds-cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the Affymetrix GeneChip® IVT Labeling Kit in the presence of biotin-labeled CTP and UTP
Hybridization protocol
the biotin-labeled IVT-RNA was fragmented. The fragmented cRNA was hybridized to the GeneChip® Chicken Genome Array at 45°C for 16 h according to the manufacturer’s instructions.
Scan protocol
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, and the intensities were determined with a GeneChip® scanner, which was controlled by the GeneChip® Operating Software (GCOS; Affymetrix).
Description
1. The male and female brain samples by genetic sexing were pooled and homogenized. Pooled samples ranged from 5 to 8 embryos. Specifically, 4g of total RNA from the pooled samples was used for labeling. Probe synthesis from total RNA samples, hybridization, detection, and scanning were performed according to standard protocols from Affymetrix 2. The quality of the array image was assessed as described in the Affymetrix GeneChip® expression analysis manual
Data processing
All arrays were processed using a robust multi-array average (Irizarry RA et al., 2003) using the R package Affy (Gautier L et al., 2004). Expression values were computed in detail from raw CEL files by applying the RMA model of the probe-specific correction of perfect-match probes. These corrected probe values were then subject to quantile normalization, and a median polish was applied to compute one expression measure from all probe values. The resulting RMA expression values were log2-transformed. The individual gene expression levels of male and female brains at stage 29 were compared using the unpaired Student’s t-test, and Benjamini-Hochberg correction for the false discovery rate (FDR) was used for all probe-level normalized data. For identification of differentially expressed genes, we selected genes with an FDR-adjusted p-value < 0.01 using an unpaired Student’s t-test and a fold-change of greater than 2 (absolute log fold change > 1).
