Robust-LongSAGE (RL-SAGE): An Improved LongSAGE Method for High-Throughput Transcriptome Analysis

Serial analysis of gene expression (SAGE) is a powerful technique for large-scale transcriptome analysis in eukaryotes. However, technical difficulties in the SAGE library construction, such as low concatemer cloning efficiency, small concatemer size, and a high level of empty clones, has prohibited its widespread use as a routine technique for expression profiling in many laboratories. We recently improved the LongSAGE library construction method considerably and developed a modified version called Robust-LongSAGE, or RL-SAGE. In RL-SAGE, concatemer cloning efficiency and clone insert size were increased significantly. About 20 PCR reactions are sufficient to make a library with more than 150,000 clones. Using RL-SAGE, we have made 10 libraries of rice, maize, and the rice blast fungus Magnaporthe grisea .