Gene expression analysis of glycosylation-related genes by real-time polymerase chain reaction.

Glycan molecules covalently linked to proteins or lipids control vital properties of cells, such as signaling, adherence, and migration through the body. The biosynthesis of such glycans depends on the concerted action of many endoplasmic reticulum and Golgi enzymes, a process that is tightly ordered and regulated. To understand the function of glycoconjugates in cellular interactions, it is crucial to investigate the regulation of expression of the genes encoding the "glycosylation-related" genes, encompassing large families of glycosyltransferases, glycosidases, and sulfotransferases. This chapter describes an easy, flexible, and reliable method of quantitative real-time polymerase chain reaction to measure the expression levels of 80 human glycosylation-related genes that primarily encode common enzymes involved in N- and O-linked protein glycosylation and/or glycolipids. Designing and including additional primer sets to detect more genes can easily extend the system. In order to allow the normalization of gene expression data obtained by real-time polymerase chain reaction within different cells, tissues, or under different experimental conditions, a protocol is included to detect genes suitable for use as endogenous reference genes.