Crawford, AC, White, JF, Bundock, PC, Cordeiro, GM, McIntosh, SR, Pacey-Miller, T, Rooke, L & Henry, RJ 2005, 'Cost effective SAGE libraries for wheat and barley', paper presented to the Plant and Animal Genomes XIII Conference, San Diego, California, USA, 15-19 January.
Serial analysis of gene expression (SAGE) is rapidly becoming a popular technique for transcript identification and quantification in plant DNA systems. SAGE methods have advanced recently with the development of LongSAGE, which enables more efficient transcript identification via generation of longer tags (Saha et al. 2002). Robust-LongSAGE (RL-SAGE) is a derivative of LongSAGE that has further potential to reduce sequencing costs by improving concatemer cloning efficiency (Gowda et al., 2004). Cloning efficiency is enhanced primarily through partial digestion of concatemers, which presumably linearises circular fragments. The quantity of PCR material used throughout this procedure however, is critical to the successful formation of linear high molecular weight fragments. We have produced several SAGE libraries from developing and germinating wheat and barley grains, using different starting quantities of PCR product. Optimal results were obtained with approximately 40 µg of PCR product, which yielded libraries with an average insert size of approximately 800 bp. With only minor adjustments to existing commercial protocols, these results will assist in the generation of cost-effective SAGE libraries