DOI link for Overrepresentation Analysis
Overrepresentation Analysis book
The practical importance of the Gene Ontology (GO) is linked to the fact that it has been used to annotate millions of gene products from thousands of organisms. The growth of GO parallels that of high-throughput technologies in molecular biology that allow essentially all genes in the genome to be measured experimentally. DNA microarrays consist of thousands of probes of either short oligonucleotides (roughly 25 to 60 nucleotides long) or longer cDNA probes that are complementary to the sequences to be measured. A cDNA or cRNA sample is hybridized to the probes on the microarray to detect and quantify the amount of the corresponding sequences in the sample. For gene expression profiling, the expression levels (mRNA concentrations) of thousands of genes are measured simultaneously. Often, microarray experiments are used to compare gene expression profiles under two or more biological conditions, say a comparison between healthy and diseased tissue or at different developmental stages. A typical experiment involves three or four replicate microarray experiments for each biological condition, and the statistical analysis would involve performing a t test or variant thereof on each of the genes on the microarray . More recently, next-generation sequencing methodologies including especially RNA-seq  are further extending the range of transcription profiling experiments that can be performed. The outcome of transcription profiling experiments, whether by microarray or RNAseq, is often a list of hundreds or even thousands of differentially expressed genes.