Serial analysis of cereals

Serial Analysis of Gene Expression (SAGE) is a technique that allows genome-wide expression studies based on the sequencing of unique gene tags. This method produces a comprehensive and quantitative picture of an RNA transcript population. The principle that a short nucleotide sequence, or tag, can effectively identify the original gene transcript from whence it came, allows these tags to be linked together for rapid sequencing. The tags are extracted from the raw sequence and the frequency of a particular tag provides a quantitative measure of its corresponding gene expression. Recent improvements to the conventional SAGE method have allowed the generation of longer 21bp tags to enable more effective transcript identification (Saha et al. 2002). An improvement to the efficiency of concatemer cloning, through partial digestion of concatemers, has the potential to further reduce sequencing costs (Gowda et al. 2004). Previously, SAGE libraries were constructed with no quantitative measure of PCR product. By producing SAGE libraries using different starting quantities of PCR product we have identified an optimal concentration for the successful formation of linear high molecular weight concatemers. The latest advancements mean that SAGE may be the most useful high-throughput approach for gene profiling in terms of scale, economy and sensitivity. We have produced SAGE libraries from developing wheat and germinating barley grains. The resulting gene expression profiles will provide an insight into the biological and physiological activity of cereal grains.