Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis

Abstract

The serial analysis of gene expression (SAGE) technique is an important tool for genome-wide gene expression analysis. However, the requirement of a large amount of mRNA for the analysis and the difficulties in generating high-quality tag and ditag fragments for the construction of a SAGE library often interfere with the successful performance of the SAGE technique. We developed two procedures to solve these issues: (i) introducing low-cycle PCR amplification of the 3′ cDNA before the BsmFI digestion of the 3′ cDNAs and (ii) gel purifying the BsmFI-released tag fragments before ditag formation. These modifications provide a large quantity of initial 3′ cDNAs and high-quality tags and ditags for the construction of SAGE libraries.