In the technique, mRNA is isolated, copied into cDNA, tagged at the 3' end with biotin, and then cut with a restriction enzyme. The tagged fragments are then isolated with streptavidin-coated beads, amplified, ligated into a single molecule, and sequenced. The enumeration of each "tag" indicates the level of expression of the gene it represents. The technique has several advantages over microarray analysis, as it can detect genes with no prior sequence information, is sensitive for low-abundance genes, and provides digital quantification of gene expression. The public database SAGEmap (www.ncbi.nlm.nih.gov/sage) is available to compare data with known gene expression profiles.
Until recently, researchers using the SAGE technique have had to follow a complex and labor-intensive protocol involving specialized buffers that were not commercially available. "A recent survey suggested that 50 percent of individuals who try SAGE technology from 'scratch' fail," says Amy Duncan, product manager at Invitrogen. "Problems may arise ...
