Samples were decapitated at day 18, and tissue was extracted
Extracted molecule
total RNA
Extraction protocol
Heart, left gonads and diencephalon from each of four male and four female embryos were homogenized by syringe and needle followed by use of the Homogenizer system (Invitrogen, Carlsbad, CA, USA). RNA was extracted from heart and brain homogenates with the PureLink Micro-to-Midi Total RNA Purification System (Invitrogen), and RNA was then DNase treated with a DNA-free kit (Ambion, Austin, TX, USA). Due to limited amounts of available tissue, gonad RNA extraction was performed with an RNeasy Micro Kit (Qiagen, Hilden, Germany) with integrated DNase treatment. RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc, Palo Alto, CA). Two micrograms of total RNA from each sample were used to prepare biotinylated fragmented cRNA according to the GeneChip® Expression Analysis Technical Manual (Rev. 5, Affymetrix Inc., Santa Clara, CA). Affymetrix GeneChip® expression arrays (Human Genome U133 Plus 2.0 Array) were hybridized for 16 hours in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Analysis Technical Manual (Rev. 5, Affymetrix Inc., Santa Clara, CA), the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChip® Scanner 3000 7G. In total, 24 hybridizations were made (4 individuals x 3 tissues x 2 sexes), however, three samples failed to meet Affymetrix quality control criteria and were removed from further analysis (heart and brain from one male, and gonads from one female).
Label
biotin
Label protocol
affyemtrix protocol
Hybridization protocol
affymetrix protocol
Scan protocol
affymetix protocol
Description
See Ellegren et al. in Press
Data processing
All pre-processing and statistical analysis of microarray data was performed in R version 2.4.1 using Bioconductor packages release 1.9 . The CEL files were processed using GCRMA30, a background adjustment method taking into account the GC content of probes when assessing non-specific binding, followed by quantile normalization and median-polish summarization of probe intensities into probe set intensities. A linear model was fitted to the log2 of the expression levels based on all probe sets and considering sex and tissue as a combined factor using the Lim,ma package. After pre-processing and linear model fitting the probe sets were filtered on expression; an expression threshold was set on both average expression level and absent/present calls from the R implementation of the Affymetrix MAS 5.0 algorithm. Only probe sets with average expression over a defined threshold and present in more than half of the samples within at least one tissue-sex combination were considered as significantly expressed. This resulted in 15,398 probe sets for heart, 16,846 for brain and 17,438 for gonads, and these probe sets composed the reference for analysis of differently expressed genes between the sexes. Annotation of probe sets. Annotations for the probe sets were extracted from Ensembl (www.ensembl.org) via biomRt in R. The Ensembl mapping of probe sets is based on alignments of individual probes to the chicken genome version 2.1 (WASHUC2 May 2006) and covers 21,885 of the 37,693 chicken-specific probe sets, which is close to the total number of protein-coding genes in the chicken genome identified by Ensembl. Several transcripts are represented by more than one probe set; the 21,885 probe sets with annotation corresponds to 14,414 unique transcripts. The genomic location for probe sets was taken from Ensembl. Gene ontology (GO) terms were available from the Gene Ontology Annotation Database (http://www.ebi.ac.uk/GOA/) for 18,239 of the 21,885 chicken probe sets with Ensembl annotation. To get a broader overview of the GO terms assigned to genes, the terms were traced back to the ancestral term at different levels in each of the gene ontology classes biological process, molecular function and cellular component. These limited sets of terms where then used to test for over- and under representation among biased genes.
