Figure 1.

Overview of differences between SAGE and SADE. (Left) Typical yields of different steps used for construction of a SAGE library. Total RNAs are extracted by using the acid guanidinium thiocyanate-phenol-chloroform method (8), and poly(A) RNAs are isolated by oligo(dT)-cellulose chromatography. Synthesis of cDNA is initiated with a biotinylated oligo(dT) primer. The cDNA then is cleaved with the SAGE anchoring enzyme (Sau3AI), and its 3′-end is isolated by binding to streptavidin beads. After recovery and ligation of cDNA tags to form ditags, PCR generates the expected fragments (110 bp), together with shorter parasitic products. (Right) mRNAs are directly isolated from the tissue lysate by binding to oligo(dT) covalently bound to magnetic beads. Synthesis and cleavage of cDNA then are performed on beads. The high yield of the procedure makes it possible to generate sufficient ditags for predominant amplification of the expected product (see text for details).