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Which genes are differentially expressed in my microarray data? Are these genes enriched for certain biological pathways?

This recipe provides an outline of one method to identify known biological functions for genes that are differentially expressed between two conditions or phenotypes, using microarray data. An example use of this recipe is a case where an investigator may want to determine if a specific cancer phenotype is associated with expression of certain pathways.

 

Given a set of differentially expressed genes, the goal is to infer which biological functions (for example, Gene Ontology biological processes) are overrepresented in the set of reference genes found to be differentially expressed. In particular, this recipe uses a gene expression dataset which has two conditions: normal and mild hyperthermia. Then, GenePattern is used to identify differentially expressed genes, and finally MSigDB is used to identify biological functions and pathways that are enriched in the gene set.

Why differential expression analysis? We assume that most genes are not expressed all the time, but rather are expressed in specific tissues, stages of development, or under certain conditions. Genes which are expressed in one condition, such as cancerous tissue, are said to be differentially expressed when compared to normal conditions. To identify which genes change in response to specific conditions (e.g. cancer), we must filter or process the dataset to remove genes which are not informative.

Why perform functional annotation? Many analyses end with the retrieval of a gene list, e.g. gene expression analysis identifies a list of genes which are differentially expressed when comparing multiple conditions. However, often times a researcher has additional questions about the function or relatedness of genes in a gene list: Are the genes a part of the same pathway? Do the gene products interact physically? Do the gene products localize to a specific part of the cell? Are the genes only expressed during a certain stage of development? These questions, and others like them, can be answered by performing functional annotation on gene lists, to better understand the underlying connections between genes.

How are SNP-related genes regulated in an expression dataset? Are these genes enriched for particular biological functions?

This recipe provides one method for identifying enriched biological functions in single-nucleotide polymorphisms (SNPs). An example use of this recipe is a case where an investigator may complete a genome-wide association study (GWAS) and wants to know the SNPs that are associated with certain genomic coordinates, in order to determine which genes have particular biological functions.

In this particular example, we imagine a scenario in which an investigator completes a GWAS study, obtaining a list of genomic coordinates that are associated with SNPs. However, simply knowing these genomic coordinates is not always informative; the investigator is also interested in knowing which genes lie in these regions, and what kinds of biological functions these genes may have. In this particular example, we are interested in answering two questions:

  1. Are the genes near these SNPs enriched with particular biological functions?
  2. Are the genes near these SNPs significantly up- or down-regulated in some other biological conditions, such as cancer?

To answer the first question, we will find Gene Ontology functional annotations using Genomica. To answer the second question, we will use the Gene Set Enrichment Analysis (GSEA) module in GenePattern, comparing the SNP-associated genes to a gene expression dataset from a study of epithelial cancer stem cells. This study evaluated the ability of oncogenes to activate an embryonic stem cell program in differentiated adult tumor cells, by transforming human keratinocytes into squamous cell carcinomas using oncogenic Ras and IκBα, plus one of three genes: c-Myc, E2F3, and GFP (Wong et al. 2009. Cell Stem Cell). Comparisons between these three genes showed that c-Myc could re-activate the embroynic stem cell program. Comparing the SNPs to this gene expression dataset can determine whether this set of SNP-associated genes are differentially regulated in c-Myc samples, when compared to other genes such as GFP and E2F3.

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